Streptococcus sanguinis Class Ib Ribonucleotide Reductase

Background: Class Ib ribonucleotide reductase (RNR) is an essential enzyme for aerobic growth of S. sanguinis.

Results: Its manganese form is 3.5-fold more active than the iron form when assayed with NrdH and thioredoxin reductase.

Conclusion: This specific activity is the highest reported to date for the class Ib RNR.

Significance: Our studies suggest why manganese is important in streptococcal pathogenesis.

Abstract

Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn^III₂-Y^•) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe^II and O₂ can self-assemble a diferric-tyrosyl radical (Fe^III₂-Y^•) cofactor (1.2 Y^•/β₂) and with the help of NrdI can assemble a Mn^III₂-Y^• cofactor (0.9 Y^•/β₂). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdI_ox and Mn^II₂-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μm) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.

Introduction

Ribonucleotide reductases (RNRs)⁵ catalyze the conversion of nucleotides to deoxynucleotides, providing the monomeric precursors required for DNA replication and repair in all organisms (1). All RNRs rely on a metallo-cofactor to oxidize a conserved cysteine in the active site of the enzyme into a thiyl radical, which then initiates reduction of nucleotides (2). In the case of the class I RNRs, two subunits are required, The β subunit contains a dinuclear metallo-cofactor that oxidizes this cysteine in the α subunit where nucleotide reduction occurs. The oxidation is reversible and occurs over a 35-Å distance (3). Recently, two new cofactors of the class I RNRs have been characterized, which has led to their subclassification (Ia, Ib, and Ic). The class Ia RNRs use a Fe^III₂-Y^• cofactor in vitro and in vivo. In contrast, the class Ib RNRs are active with both Mn^III₂-Y^• and Fe^III₂-Y^• (4–8) cofactors, although the class Ic RNRs require a Mn^IVFe^III cofactor (9).

In most organisms, the genes for the class Ib RNRs are found in operons. The subunits for these RNRs, α and β, are designated NrdE and NrdF, respectively. Two additional gene products are often found within this operon as follows: NrdI, a flavodoxin that we have recently shown behaves as a flavin oxidase (10), and NrdH, a glutaredoxin-like thioredoxin (11, 12). NrdF has been known for some time to be able to self-assemble an active Fe^III₂-Y^• cofactor from apo-NrdF, Fe^II, and O₂ in a manner analogous to the corresponding β (NrdB) in the class Ia RNRs (13, 14). We have recently demonstrated with the Escherichia coli and Bacillus subtilis NrdFs (4, 5), as have Sjöberg and co-workers (8) with Bacillus anthracis NrdF, that a Mn^III₂-Y^• cofactor can be assembled from a Mn^II₂-NrdF, but only in the presence of the reduced form of NrdI (NrdI_hq) and O₂. An x-ray structure of a complex of E. coli NrdI_hq and Mn^II₂-NrdF, in conjunction with biochemical studies, recently demonstrated that the oxidant produced by NrdI_hq is channeled directly to one of the manganese ions in the Mn^II₂-NrdF (15). Recent mechanistic studies on the B. subtilis Mn^III₂-Y^• cofactor assembly further established that O₂^⨪ is the oxidant required to convert Mn^II₂-NrdF to the Mn^III₂-Y^• (16).

In many, but not all, of the class Ib RNRs, NrdH can re-reduce the disulfide in α produced concomitant with dNDP formation (Fig. 1) and thus is required for multiple turnovers (6, 11, 17–19). NrdH itself can be re-reduced in vitro by TrxR and NADPH in E. coli, B. anthracis, Staphylococcus aureus, and Corynebacterium glutamicum (6, 11, 20, 21).

FIGURE 1.

RNR catalyzes the conversion of NDPs into dNDPs concomitant with disulfide bond formation. In the S. sanguinis class Ib RNR, the reducing equivalents to re-reduce this disulfide in vivo are supplied by NrdH/TrxR/NADPH.

Identification of the metal cofactor (manganese and/or iron) in class Ib RNR in vivo under different growth conditions is an active area of investigation. Recently, Corynebacterium ammoniagenes, E. coli, and B. subtilis NrdFs isolated from their endogenous host organisms were all shown to contain a Mn^III₂-Y^• cofactor (5, 7, 22, 23). Human and most eukaryotic RNRs utilize class Ia RNRs exclusively, whereas pathogenic organisms, including Streptococcus sanguinis, Streptococcus pneumoniae, B. anthracis, S. aureus, and Corynebacterium diphtheriae depend on class Ib RNR for aerobic production of deoxynucleotides. Knowledge of how the biosynthetic pathways for the class Ia and class Ib RNRs differ could thus potentially lead to new targets for antimicrobial therapeutics (24).

S. sanguinis was chosen to understand the role of the manganese RNR in pathogenesis for several reasons. First, it has recently been shown in a screen for virulence factors for this organism that deletion of SsaB, a manganese transporter, severely reduces infectivity of this organism (25). Second, deletion of SodA (the manganese-dependent superoxide dismutase) does not adequately explain the reduced virulence of the ssaB mutant (26). Third, S. sanguinis contains a class Ib and a class III RNR, essential for aerobic and anaerobic growth, respectively (12). This organism can thus also serve as a model for many pathogenic organisms with class Ib RNR as the only aerobic source of deoxynucleotides (27). Fourth, based on phylogenetic analysis (28), S. sanguinis NrdF belongs to a subclass distinct from E. coli, B. subtilis, and C. ammoniagenes, and thus these studies may be informative as to whether all NrdFs utilize a Mn^III₂-Y^• cofactor.

In this study, we report the cloning, expression, and isolation of NrdE, NrdF, NrdI paralogs (NrdI, FmnI, and FmnG), NrdH, and two putative thioredoxin reductases TrxR1 and TrxR2. We establish in vitro that an active cofactor of S. sanguinis class Ib RNR can be assembled with both manganese and iron and that only NrdI, not FmnI or FmnG, is essential in Mn^III₂-Y^• cofactor formation in NrdF. The Mn^III₂-Y^•-NrdF has activity of 6.1 s⁻¹, 3.5-fold higher than for the Fe^III₂-Y^•-NrdF when assayed with NrdH and TrxR1. The Mn^III₂-Y^•-NrdF turnover number is very similar to the E. coli class Ia RNR (Fe^III₂-Y^•) (29). Finally, an animal model for infective endocarditis is available. Data in the accompanying paper (30) show that in vivo only the manganese NrdF appears to be active. Because mammalian host organisms use a Fe^III₂-Y^• for RNR activity, finding an inhibitor of NrdI/NrdF interactions required for Mn^III₂-Y^• cluster assembly could provide a new target for therapeutic intervention.

MATERIALS AND METHODS

General

All chemical reagents were purchased from Sigma, unless otherwise indicated. FMN was 73–79% FMN, <6% free riboflavin, <6% riboflavin diphosphates. FAD was 94% pure. MnCl₂·4H₂O (>98% pure, <5 ppm iron) and (NH₄)₂Fe(SO₄)₂·6H₂O (99%) were used as a source of Mn^II and Fe^II, respectively. Primers were synthesized by Integrated DNA Technologies. All restriction enzymes were from New England Biolabs. His₆-tagged SUMO protease was expressed in codon+ Rosetta cells (Novagen) from pTB145 provided by Prof. Bradley Pentelute (Chemistry Department, Massachusetts Institute of Technology). Buffers in this study are as follows: buffer A, 50 mm Hepes, 5% (v/v) glycerol, pH 7.6; buffer B, buffer A and 5 mm β-mercaptoethanol; buffer C, 50 mm sodium phosphate, 10% (v/v) glycerol, pH 7.6; buffer D, 50 mm Hepes, pH 7.6, 15 mm MgSO₄, 1 mm EDTA. O₂-saturated buffer A was prepared immediately prior to use by sparging on ice with O₂ for at least 30 min. [³H]CDP was obtained from ViTrax and diluted with CDP in buffer A to 6,000–12,000 cpm/nmol before use. Radioactive samples were analyzed using a Beckman Coulter LS6500 scintillation counter. EPR spectra were acquired on a Bruker EMX X-band spectrometer at 77 K using a finger Dewar filled with liquid nitrogen and 706-PQ Wilmad EPR tubes or at room temperature (RT) using a Wilmad flat cell (150 μl). UV-visible spectra of anaerobic samples were acquired on a Varian Cary 3 UV-visible spectrophotometer using anaerobic cuvettes (Starna) with Teflon silicon septa (Thermo Scientific) and anaerobic titrations used a 50-μl gas tight syringe with a repeat dispenser (Hamilton). Fluorescence measurements were carried out using a Photon Technology International QM-4-SE spectrofluorometer and FELIX software. Manganese concentrations were measured using a PerkinElmer Life Sciences AAnalyst 600 atomic absorption spectrometer, and Fe^II concentrations were measured by the ferrozine assay (31). DNA sequencing and MALDI-TOF mass spectrometry were performed at the Biopolymers Laboratory, Massachusetts Institute of Technology. All anaerobic procedures were carried out in a glove box (MBraun), and all proteins were purified at 4 °C. For each protein molecular mass and the following extinction coefficients (ϵ) were calculated using ExPASy: NrdF or β₂ (ϵ₂₈₀ = 133,620 m⁻¹ cm⁻¹); NrdE or α₂ (ϵ₂₈₀ = 161,140 m⁻¹ cm⁻¹); NrdH (ϵ₂₈₀ = 2,980 m⁻¹ cm⁻¹). Concentrations of NrdF and NrdE are reported for dimers. Concentration of NrdI_ox was determined based on A₄₅₁ of FMN_ox cofactor (ϵ₄₅₁ = 12,170 m⁻¹ cm⁻¹). Concentrations of TrxR1 and TrxR2 were estimated based on absorbance of bound FAD at 455 and 463 nm, respectively. As an extinction coefficient at these wavelengths, we used a number previously measured for E. coli TrxR, ϵ₄₅₆ = 11,300 m⁻¹ cm⁻¹ (32).

Cloning of the S. sanguinis Genes Required for RNR Activity

Platinum Pfx DNA polymerase (Invitrogen), sense and antisense primers (supplemental Table S1) containing restriction sites (underlined), were used to amplify nrdE, nrdF, nrdI (SSA_2263), fmnG (SSA_1668), fmnI (SSA_1683), trxR2 (SSA_0813) and trxR1 (SSA_1865) from WT S. sanguinis SK36 genomic DNA as a template. Taq DNA polymerase (Promega), sense and antisense primers without restriction sites, was used to amplify nrdH. All PCRs were performed using 60 ng of genomic DNA, 0.25 μg of each primer in a total volume of 50 μl, and a thermocycler program that was optimized for long AT-rich primers (supplemental Table S2). nrdE, nrdF, nrdI, fmnG, fmnI, trxR1, and trxR2 were cloned into pET28a (Novagen) and nrdF into pET24a via NdeI and BamHI or NdeI and XhoI restriction sites using T4 DNA ligase (Promega). nrdH was cloned into pETSUMO using T4 DNA ligase as described in the manufacturer's protocol (Invitrogen).

Expression and Purification of NrdI, FmnG, FmnI, Apo-NrdF, and NrdE

pET28a-nrdI was transformed into BL21(DE3) cells and expressed in LB in the presence of 50 μg/ml kanamycin. The culture was grown at 37 °C with shaking at 200 rpm to OD₆₀₀ = 0.5, and then riboflavin was added to a final concentration of 10 mg/liter. NrdI culture was induced at A₆₀₀ = 0.7–0.8 with isopropyl β-d-1-thiogalactopyranoside (Promega) to a final concentration of 0.4 mm and incubated at 30 °C. In 4–5 h, cells were harvested by centrifugation at 3,000 × g for 10 min at 4 °C, frozen in liquid nitrogen, and stored at −80 °C. Typical yield was 1.8 g wet cell paste/liter of culture.

Cell pellet (18 g) was resuspended in 90 ml of buffer C, containing 1.7 mm FMN and two Complete protease inhibitor mixture tablets (Roche Applied Science). The cell suspension was passed twice through a French pressure cell at 14,000 p.s.i. and then centrifuged at 30,000 × g for 20 min. Nucleic acids were precipitated by addition of streptomycin sulfate solution to a final concentration of 1.3% (w/v) with stirring for 15 min. After the solution was spun down at 30,000 × g for 30 min, the supernatant was loaded onto Ni-NTA-agarose column (Qiagen, 1.5 × 3.4 cm, 6 ml) pre-equilibrated with buffer C, and the column was washed with buffer C containing 100 mm NaCl and 20 mm imidazole, pH 7.6, until A₂₈₀ of the flow-through was <0.02. The protein was eluted with a 70 × 70-ml linear gradient of 20–250 mm imidazole in buffer C. NrdI-containing fractions, identified by A₂₈₀ and A₄₁₅, were pooled, loaded onto a Q-Sepharose Fast Flow column (GE Healthcare, 2.5 × 6.5 cm, 32 ml) pre-equilibrated with 100 mm NaCl in buffer C, and the column was washed with the same buffer. Flow-through fractions containing NrdI were pooled, and the protein was concentrated using an Amicon YM-10 centrifugal filter (Millipore), desalted on Sephadex G-25 column (Sigma, 1.5 × 36.5 cm, 64 ml), and further concentrated to 1–1.5 mm. A typical yield was 3 mg/liter of culture, and the protein was >95% homogeneous by SDS-PAGE analysis. FmnI and FmnG were purified as described above for NrdI.

NrdF and NrdE were expressed in BL21(DE3) and purified using protocols previously optimized for E. coli class Ib RNR (10). NrdF and DNA bands partially overlap (judged by A₂₆₀/A₂₈₀ and SDS-PAGE analysis); to remove DNA, the pooled NrdF fractions were chromatographed twice on a Q-Sepharose Fast Flow column.

Expression and Purification of NrdH, TrxR1, and TrxR2

pETSUMO-nrdH was transformed into BL21(DE3) cells and expressed in LB in the presence of 50 μg/ml kanamycin. The culture was grown at 37 °C with shaking at 200 rpm to OD₆₀₀ = 0.6, and then the temperature was lowered to 30 °C, and the culture was induced with isopropyl β-d-1-thiogalactopyranoside to a final concentration of 0.4 mm. In 4 h, cells were harvested by centrifugation at 3,000 × g for 10 min at 4 °C, frozen in liquid nitrogen, and stored at −80 °C. Typical yield was 2.3 g of cell paste/liter of culture.

Cell pellet (9.2 g) was resuspended in 46 ml of buffer B containing one Complete protease inhibitor mixture tablet. The cell suspension was passed twice through a French pressure cell at 14,000 p.s.i. and then centrifuged at 20,000 × g for 25 min. Nucleic acids were precipitated by the addition of a streptomycin sulfate solution to a final concentration of 0.5% (w/v) with stirring over 20 min. After the solution was spun down at 20,000 × g for 30 min, the supernatant was loaded onto Ni-NTA-agarose column (1.5 × 2.5 cm, 4.4 ml) pre-equilibrated with buffer B containing 30 mm imidazole, and the column was washed with the same buffer until A₂₈₀ of the flow-through was <0.02. The protein was eluted with 50 ml of 200 mm imidazole in buffer B, and 2-ml fractions were collected. Fractions containing SUMO-NrdH (21.6 kDa) were identified by SDS-PAGE (12% (w/v) acrylamide), pooled, and loaded onto a Q-Sepharose Fast Flow column (2.5 × 5.5 cm, 27 ml) pre-equilibrated with 100 mm NaCl in buffer B. The column was then washed with the same buffer (300 ml) then SUMO-NrdH was eluted with 100 × 100-ml linear gradient of 100–500 mm NaCl in buffer B, and 3.6-ml fractions were collected. Fractions with high A₂₈₀ were analyzed by SDS-PAGE, pooled, and concentrated to 470 μm using an Amicon YM-10 filter.

SUMO-NrdH (470 μm) was divided into 0.6-ml aliquots, and each was incubated with SUMO protease (150 μl, 50 μm) overnight at 4 °C. About 60% of the protein was cleaved under these optimized conditions. The digested SUMO-NrdH was loaded directly onto a Ni-NTA-agarose column (1.5 × 2.5 cm, 4.4 ml) pre-equilibrated with 30 mm imidazole in buffer B. The column was washed with the same buffer, and 1-ml fractions were collected. Fractions containing NrdH (8.2 kDa), assessed by SDS-PAGE analysis (16% (w/v) Tricine gel, Invitrogen), were pooled and concentrated using an Amicon YM-3 filter. NrdH (2 ml) was then loaded onto Sephadex G-25 column (1.5 × 33 cm, 58 ml) pre-equilibrated with buffer A containing 10 mm DTT and 1 mm EDTA, eluted with the same buffer, and concentrated to 1 mm using an Amicon YM-3 filter. The ratio A₂₈₀/A₂₆₀ of homogeneous NrdH is 1.3. Successful removal of the tag was confirmed by MALDI-TOF MS.

To remove DTT from NrdH, required for the DTNB assay, NrdH (400 μl, 1 mm) was loaded onto Sephadex G-25 column (1 × 6.5 cm, 5 ml) pre-equilibrated with buffer A, and 0.5-ml fractions were collected. Fractions containing NrdH, as judged by A₂₈₀/A₂₆₀, were pooled and concentrated to 450 μm as described above.

pET28a-trxR1 (or pET28a-trxR2) was transformed into BL21(DE3) cells and trxR1 (trxR2) overexpressed in the presence of 50 μg/ml kanamycin. The culture was grown at 37 °C to OD₆₀₀ = 0.5, and then riboflavin (10 mg/liter) was added, and the temperature was lowered to 30 °C; 10 min later, the culture was induced with isopropyl β-d-1-thiogalactopyranoside to a final concentration of 0.4 mm. In 4 h, the cells were harvested, as above to give typical yields of 2.5 g wet cell paste/liter of culture.

Cell pellet (7.5 g) was resuspended in 38 ml of buffer A containing a Complete protease inhibitor tablet and 1.7 mm FAD. The cell suspension was passed twice through a French pressure cell at 14,000 p.s.i. and then centrifuged at 20,000 × g for 25 min. Nucleic acids were precipitated by the addition of streptomycin sulfate solution to a final concentration of 1% (w/v). After the solution was spun down at 20,000 × g for 30 min, the supernatant was loaded onto a Ni-NTA-agarose column (1 × 4.2 cm, 3 ml), pre-equilibrated with buffer A with 30 mm imidazole, and the column was washed with the same buffer until A₂₈₀ was <0.02. The protein was eluted with 20 ml of 200 mm imidazole in buffer A. The yellow fractions were pooled, diluted 4-fold with buffer A, and loaded onto Q-Sepharose Fast Flow column (2.5 × 5.5 cm, 27 ml), pre-equilibrated with buffer A and 100 mm NaCl, and the column was washed with the same buffer (∼150 ml). TrxR1 (TrxR2) was eluted with 100 × 100-ml linear gradient of 100–550 mm NaCl in buffer A and concentrated using an Amicon YM-30 filter; NaCl was removed by further dilution/concentration in buffer A.

Optimized Cofactor Assembly from Apo-NrdF Loaded with Fe^II or Mn^II

Buffers and proteins were degassed on a Schlenk line with at least five cycles of evacuation and refilling with argon. Concentrated NrdI_ox (0.5–1 mm) and apo-NrdF (0.8 mm) were stable at 4 °C for at least 5 days. To prepare Mn^II₂-NrdF, apo-NrdF (45 μl, 800 μm) was incubated with 6 eq of Mn^II (2 mm solution, freshly prepared) in buffer A at 37 °C for 15 min. A mixture of Mn^II₂-NrdF (60 μm) and NrdI_ox (120 μm) in buffer A in a total volume of 600 μl was titrated anaerobically with dithionite (∼3 mm, standardized using potassium ferricyanide (33)) until NrdI_ox was completely reduced to NrdI_hq (judged by the disappearance of the band at 580 nm associated with NrdI_sq). Cluster assembly was initiated by bubbling O₂ through the Mn^II₂-NrdF/NrdI_hq solution for ∼1 min. This protocol typically gave 0.6 Y^•/β₂. Increased yields of Y^•/β₂ were obtained by recycling NrdI. After cluster assembly as described above, the mixture of Mn^III₂-Y^• (0.6 Y^•/β₂) and NrdI_ox was again degassed on a Schlenck line (six cycles) and transferred inside the glove box to an anaerobic cuvette. The UV-visible spectrum was recorded to ensure that radical content remained intact, and then the mixture was titrated with dithionite (∼3 mm) to reduce NrdI_ox to NrdI_hq. O₂ was then bubbled through the sample for ∼1 min. To remove free Mn^II and NrdI, the mixture was incubated with EDTA (5 mm) at 4 °C for ∼30 min and loaded onto a Q-Sepharose Fast Flow column (1 × 3 cm, 2.5 ml) pre-equilibrated with 200 mm NaCl in buffer A. The column was washed with 200 mm NaCl (buffer A, 25 ml), and Mn^III₂-Y^•-NrdF was eluted with 500 mm NaCl (buffer A, 20 ml); 1-ml fractions were collected, and the protein was concentrated using an Amicon YM-30 filter. Typical radical content was 0.9–1.0 Y^•/β₂.

For cluster assembly with iron, apo-NrdF (∼150 μl, 900 μm) was incubated anaerobically with 6 eq of Fe^II at 37 °C for 15 min and then diluted with buffer A to 60 μm. Cluster assembly was initiated by addition of an equal volume of oxygenated buffer A. To remove excess iron, ferrozine (80-fold excess over NrdF) and sodium dithionite (40-fold excess) were added, and the mixture was incubated on ice for 5 min. The protein was then desalted on a Sephadex G-25 column (1 × 11 cm, 8.5 ml) in buffer A. NrdF-containing fractions were pooled and concentrated. The amount of NrdF-bound Fe^II was measured by the ferrozine assay (31).

Y^• Quantitation

All EPR spectra used for spin quantitation were acquired under nonsaturating conditions at 77 K (4). Spin quantitation was performed by double integration of the signal and comparison with a standard of E. coli Fe^III₂-Y^•-NrdB.

K_d for the NrdI_hq and Mn^II₂-NrdF Interaction

The procedure is a minor modification of that previously reported (16). Under anaerobic conditions at 23 °C, NrdI_hq (240 or 360 μm, ∼ 40 μl) in buffer C was added in 2-μl portions using an air-tight Hamilton syringe into Mn^II₂-NrdF (1 or 3 μm, 700 μl) in the same buffer. To ensure that NrdI_hq remains reduced throughout titration, buffer C also contained 100 μm dithionite. After each injection, the cuvette was inverted several times, and the solution was allowed to equilibrate at RT in the dark for 1 min, and the spectrum was recorded. The excitation wavelength was 380 nm, and the emission was measured from 475 to 625 nm in 1 nm steps with a 1-s integration time. The excitation and emission bandwidth slit was 1.5 and 0.75 mm, respectively. No photobleaching was detected using these settings. Data were analyzed by the method of Eftink (34). The titration was performed four times using different concentrations of Mn^II₂-NrdF and NrdI_hq, and the data were fit in IgorPro to obtain the stoichiometry of NrdI_hq binding to NrdF (n) and the dissociation constant (K_d).

General Crystallographic Methods

All crystallographic datasets were collected at the Life Sciences Collaborative Access Team (LS-CAT) or General Medical Sciences and Cancer Institutes Collaborative Access Team (GM/CA-CAT) beamlines at the Advanced Photon Source and processed using the HKL2000 software package (35). Iterative rounds of refinement and model building were performed using Refmac5 (36) and Coot (37). Ramachandran plots were calculated with Molprobity (38) and figures were generated with the PyMOL Molecular Graphics System (Schrödinger, LLC). Internal channel calculations were performed with HOLLOW (39) using a 1.4 Å probe radius. Electrostatic surface potential calculations were carried out using the PyMOL APBS plugin (40). Electron density maps were calculated with FFT (41). Table 1 reports all data collection and refinement statistics.

TABLE 1.

Data collection and refinement statistics for the S. sanguinis Mn^II₂-NrdF and NrdI_ox x-ray structures

	S. sanguinis MnII2-NrdF	S. sanguinis NrdIox
Data collection
Space group	P21	C2
Resolution	2.65 Å (2.70 to 2.65 Å)	1.88 Å (1.91 to 1.88 Å)
Rsyma,b	0.166 (0.747)	0.084 (0.705)
〈I/σI〉	10.8 (2.2)	16.9 (2.4)
Completeness	99.9% (98.7%)	100% (100%)
Refinement
Rworkc/Rfreed	0.237/0.276	0.207/0.238
Average B-factor	28.7	24.2
Root mean square deviations
Bond lengths	0.006 Å	0.005 Å
Bond angles	0.849°	1.008°

^a R_sym = Σ|I_obs − I_av|/ΣF_obs, where the summation is over all reflections.

^b Values in parentheses refer to the highest resolution shell.

^c R_work = Σ|F_obs − F_calc|/ΣF_obs.

^d For calculation of R_free, 5% of the reflections was reserved.

X-ray Structure Determination of S. sanguinis Mn^II₂-NrdF

Crystals of S. sanguinis Mn^II₂-NrdF (25 mg/ml in 20 mm Hepes buffer, 5% (v/v) glycerol, pH 7.6) were generated using the hanging drop vapor diffusion method with 25% (w/v) PEG 3000, 250 mm magnesium formate, and 100 mm Hepes, pH 7.6, as the precipitating solution. Crystals appeared after 2 weeks of incubation at RT and were prepared for data collection by mounting on rayon loops and flash freezing in liquid nitrogen following cryoprotection by brief soaking in well solution containing 25% (v/v) glycerol. X-ray diffraction datasets were processed as described above with additional scaling performed using the UCLA MBI Diffraction Anisotropy Server (42). The structure was solved by molecular replacement using BALBES (43) with the Salmonella typhimurium Fe^III₂-NrdF structure (PDB accession code 1R2F) as the search model. Eight copies of NrdF, arranged into four β2 dimers, are present in the asymmetric unit. The quality of the electron density map varies widely between the eight monomers. The electron density is the least well defined for two of the monomers (chains A and G) and is of the highest quality for chains B and H. The latter subunits were used to draw conclusions about the structural features of the metal-binding site and oxidant channel. To aid in model building, tight noncrystallographic symmetry restraints were used in the initial phases of model refinement and released in the final rounds. The final model consists of residues 3–287 for chain A, residues 3–286 for chains B–F, residues 4–286 for chains G and H, two Mn^II ions per NrdF monomer, and 64 water molecules. Ramachandran plots show that 99.9% of residues are in allowed and generously allowed regions.

X-ray Structure Determination of S. sanguinis NrdI_ox

Crystals of S. sanguinis NrdI_ox (25 mg/ml in 20 mm Hepes buffer, 5% (v/v) glycerol, pH 7.6) were generated from a commercial screen (Qiagen) using the sitting drop vapor diffusion method with 30% (w/v) PEG 4000, 200 mm ammonium sulfate, and 100 mm sodium acetate, pH 5.6 as the precipitating solution. Crystals appeared after 1 week of incubation at RT and were prepared for data collection by addition of a well solution, in a 1:1 ratio, containing 50% (v/v) glycerol to the crystallization drop followed by mounting on rayon loops and flash freezing in liquid nitrogen. The structure was solved by molecular replacement using PHASER (44) with B. subtilis NrdI_ox (PDB accession code 1RLJ) as the search model. Ramachandran plots indicate that 100% of residues are in allowed and generously allowed regions. The asymmetric unit contains two NrdI monomers and the final model consists of residues 2–66, 72–154 in chain A, residues 2–154 in chain B, two FMN molecules, two sulfate molecules, and 204 water molecules. Residues 67–71 in chain A are disordered and could not be modeled. Attempts to determine the structure of reduced forms of NrdI by soaking NrdI_ox crystals in 10–100 mm solutions of dithionite produced color changes in the crystals, but structures obtained from the resulting diffraction datasets did not exhibit any significant conformational changes in response to FMN reduction.

DTNB Assay for TrxR1/TrxR2

In a final volume of 290 μl NADPH (300 μm), variable amounts of DTT-free NrdH (0.06–5 μm), 100 mm Tris (pH 7.5 at 20 °C), and 2 mm EDTA were mixed. DTNB was added to a final concentration of 1 mm, and the mixture was equilibrated to 25 °C in a cuvette. The reaction was initiated by addition of TrxR1 (17.5 nm/dimer) and monitored by change in A₄₁₂. The turnover number was calculated as described previously (45). A similar experiment was carried out with TrxR2 (17.5 nm/dimer) and NrdH (5-30 μm); no change in A₄₁₂ was observed.

Activity Assays

Three sets of conditions optimized to assay S. sanguinis RNR using DTT, NrdH/DTT, or NrdH/TrxR1/NADPH as the reductant are described. 1) For DTT a typical activity assay contained in a final volume of 170 μl the following: reconstituted NrdF (0.2 μm), NrdE (2 μm), dATP (100 μm), DTT (20 mm), [³H]CDP (0.5 mm, 11,700 cpm/nmol) in buffer D at 37 °C. Aliquots (30 μl) were removed at 0, 3, 6, 9, and 12 min, and the reaction was stopped by heating at 100 °C for 2 min. 2) For NrdH/DTT, a typical assay contained in a final volume of 170 μl the following: NrdF (0.07 μm), NrdE (0.14 μm), dATP (100 μm), DTT (20 mm), NrdH (10 μm), [³H]CDP (0.5 mm, 6,644 cpm/nmol) in buffer D at 37 °C, and aliquots (30 μl) were taken at 0, 1, 2, 3, 4 min. 3) For NrdH/TrxR1/NADPH, a typical assay contained in a final volume of 170 μl the following: NrdF (0.07 μm), NrdE (0.14 μm), dATP (100 μm), NrdH (DTT-free, 10 μm), TrxR1 (0.5 μm), NADPH (1 mm), [³H]CDP (0.5 mm, 6,644 cpm/nmol) in buffer D at 37 °C, and aliquots (30 μl) were taken at 0, 1, 2, 3, 4 min. [³H]dCDP was quantitated by the method of Steeper and Steuart (46). One unit of activity is defined as the production of 1 nmol of dCDP per min.

K_m for NrdE-NrdF Interaction (47)

The reactions contained in a final volume of 170 μl the following: dATP (100 μm), DTT (20 mm), NrdH (10 μm), [³H]CDP (0.5 mm), and NrdF/NrdE in a ratio 1:1 (1 to 80 nm) in buffer D at 37 °C. Protein solutions below 0.1 μm also contained BSA (0.2 mg/ml). Aliquots (30 μl) were taken over 12 min for samples containing 1–5 nm, over 8 min for samples with 7–10 nm, and over 4 min for samples containing 20–80 nm NrdF and NrdE.

RESULTS

Identification of the Genes for Cluster Assembly and Activity of S. sanguinis RNR

The genome of S. sanguinis SK36 has been sequenced, and nrdH-nrdE-nrdK-nrdF were annotated in a single operon (48). A search for nrdI using B. subtilis nrdI as the query sequence revealed three candidate genes: SSA_2263 (nrdI), SSA_1668 (fmnG), and SSA_1683 (fmnI) (Fig. 2A). A general screen for genes essential under aerobic conditions identified only SSA_2263 (49). Thus, SSA_2263 was tentatively annotated as NrdI. Further analysis of this nrdI sequence, specifically the spacing between the ribosomal binding site and start codon, and ClustalW sequence alignments of characterized NrdIs suggested that Met⁵ is the actual start site and that the annotated start site is incorrect (Fig. 2B and supplemental Fig. S1). SSA_1668 and SSA_1683 were shown to bind FMN, and their genes were named fmnG and fmnI, respectively. Finally, to identify candidates for TrxR, B. anthracis thioredoxin reductase (BA5387) was chosen for a BLAST search, and two candidate genes, SSA_1865 or trxR1 (TrxR1) and SSA_0813 or trxR2 (TrxR2), were identified. Studies in the accompanying paper (30) reveal that only SSA_1865 is essential under aerobic growth conditions.

FIGURE 2.

Location and organization of the genes of S. sanguinis associated with the class Ib RNR and its metallo-cofactor assembly and the identification of the nrdI start codon and ribosomal binding site. A, three annotated NrdIs and the RNR operon containing the genes for the redoxin that re-reduces the active site disulfide (nrdH), the two subunits of RNR (nrdE and nrdF), and an unknown open reading frame nrdK. B, two possible start codons for NrdI. Sequences upstream of the putative nrdI start codons are in black, the start codons and nrdI are in red. RBS, ribosomal binding site.

Expression and Purification of Apo-NrdF, NrdE, NrdI, FmnI, FmnG, NrdH, and TrxRs

Genes encoding NrdE, NrdF, NrdI, FmnI, FmnG, NrdH, TrxR1, and TrxR2 were amplified by PCR using S. sanguinis SK36 genomic DNA as a template. The gene for each protein was cloned into pET vectors for expression in BL21(DE3), and the sequences were verified. Apo-NrdF was expressed without a tag in pET24a. NrdE, NrdI, FmnI, FmnG, TrxR1, and TrxR2 all were expressed in pET28a with an N-terminal His₆ tag and a 10-amino acid linker (MGSSHHHHHHSSGLVPRGSH). To obtain high yields of soluble protein, NrdH was cloned into pETSUMO and expressed as a fusion with a His₆-SUMO tag, and subsequent to protein purification, the His₆-SUMO tag was removed using SUMO protease. All of the proteins were purified to >95% homogeneity by conventional methods (Ni-NTA and ion exchange chromatography).

Characterization of NrdI

Phylogenetic analysis suggests that S. sanguinis NrdI is likely distinct from previously characterized NrdIs (5, 10, 16). Thus, NrdI (full-length and truncated by four amino acids, Fig. 2B) along with FmnI and FmnG were expressed and purified. As discussed subsequently, neither FmnI nor FmnG support Mn^III₂-Y^• assembly in NrdF, whereas both the full-length and truncated NrdIs support Mn^III₂-Y^• assembly in NrdF to the same extent. Given our re-annotation of NrdI, the truncated NrdI was used in subsequent experiments.

The characterization of S. sanguinis NrdI followed our recent protocols for E. coli and B. subtilis NrdIs (5, 10). NrdI as isolated contained 0.8 FMN/NrdI (A₂₇₄/A₄₅₁ = 3.4), and the extinction coefficient (ϵ_451ox) of NrdI-bound FMN was calculated to be 12.17 mm⁻¹ cm⁻¹. The amount of FMN semiquinone was previously shown to be a distinguishing factor between generic flavodoxins and the NrdIs. Although flavodoxins have very distinct half-reaction reduction potentials, allowing accumulation of 100% semiquinone upon FMN reduction, the NrdIs have similar half-reaction reduction potentials. To determine the extent of semiquinone formation, anaerobic titration of NrdI_ox with dithionite was performed (data not shown). The results indicate that S. sanguinis NrdI stabilizes 35% of a neutral semiquinone FMN (ϵ_580sq = 5.3 mm⁻¹ cm⁻¹, Fig. 3A) at RT, similar to E. coli (30%) (4) and B. subtilis (24%) (5) but distinct from B. cereus (100%) (50) and B. anthracis (60%) (51) NrdIs.

FIGURE 3.

Spectroscopic characterization of S. sanguinis NrdI, Mn^II₂-NrdF, and assembled clusters. A, spectra of Nrd_Iox (red), NrdIhq (black), and Nrd_Isq (blue) in the presence (dotted) or absence (solid) of NrdF in buffer C. B, comparison of the EPR spectra (77 K) of S. sanguinis Fe^III₂-Y^•-NrdF (1.2 Y^•/β₂, acquired at 50 microwatt power), and Mn^III₂-Y^•-NrdF (0.9 Y^•/β₂, 1 milliwatt power). Spectra were normalized for NrdF concentration and power. Other spectrometer settings were 9.45 GHz, 1.5 G modulation amplitude, and 10.24 ms time constant. C, EPR spectrum of Mn^II₂-NrdF (380 μm) at 9 K, 0.1 milliwatt. To prepare Mn^II₂-NrdF, apo-NrdF (210 μl, 900 μm) was incubated with 6 eq of Mn^II in buffer A at 37 °C for 15 min. Unbound Mn^II was removed using a Sephadex G-25 column (36.5 × 1.5 cm, 64 ml) in buffer A, and the protein was concentrated on an Amicon YM-30 filter. The resulting protein contained 3.8 Mn^III/β₂. D, visible spectra of line 1, 100 μm NrdI_ox (dotted line); line 2, sample prepared by mixing 100 μm NrdI_hq, 50 μm Mn^II₂-NrdF, and O₂; and line 3, sample resulting from titration of line 2 with dithionite to re-reduce NrdI_ox to NrdI_hq followed by addition of O₂. All samples are in buffer A.

Assembly of an Active NrdF Cofactor with Iron and Manganese

For Fe^III₂-Y^•, apo-NrdF was incubated anaerobically with Fe^II for 15 min at 37 °C followed by O₂ addition. The spectrum of the resulting product revealed a sharp feature at 408 nm and broad bands at 325 and 370 nm corresponding to the Y^• (1.2 Y^•/β₂) and the diferric cluster, respectively. The EPR spectrum of Fe^III₂-Y^• is similar to those previously reported (Fig. 3B, red) for E. coli, C. ammoniagenes, and S. typhimurium (4, 14, 52).

Mn^III₂-Y^•

Apo-NrdF was mixed aerobically with Mn^II (6 eq), incubated at 37 °C for 15 min, and the free Mn^II removed by Sephadex G-25 chromatography. Atomic absorption analysis typically gave 3.6–3.8 Mn^II per β₂. The EPR spectrum of Mn^II₂-NrdF at 9 K (Fig. 3C) is very similar to E. coli and S. typhimurium Mn^II₂-NrdFs and distinct from the B. subtilis Mn^II₂-NrdF (4, 14, 16).

Typically in cluster assembly studies, Mn^II₂-NrdF is generated as above and incubated anaerobically with NrdI_hq, without Mn^II removal, followed by exposure to O₂. The UV-visible spectrum of the resulting NrdF (Fig. 3D, line 2) reveals a typical Y^• feature at 408 nm and absorption features at 550 and 330 nm associated with the Mn^III₂ cluster. Quantitative analysis typically gives 0.5–0.6 Y^•/β₂ and 3.1–3.4 Mn^III/β₂. Many variables (pH, temperature, and ratio of manganese/NrdF) were examined in an effort to increase the amount of Y^•/β₂; all conditions resulted in similar yields. A higher yield of Y^•/β₂, however, was achieved by recycling the NrdI. The NrdI_ox-NrdF mixture resulting from the first effort to assemble cluster (Fig. 3D, line 2) was made anaerobic and titrated with dithionite to reduce NrdI_ox into NrdI_hq. The sample was then re-exposed to O₂ (Fig. 3D, line 3). This recycling process typically gives 0.9 Y^•/β₂ and 3.7 Mn^III/β₂. The EPR spectrum of the Mn^III₂-Y^• at 77 K is a broad signal with a line width of 150 G and is shown in Fig. 3B (black line). Furthermore, as in the case of the other Mn^III₂-Y^•-NrdFs (4, 5, 22), the spectrum dramatically sharpens at 4 K (data not shown). Attempts to assemble Mn^III₂-Y^• cluster using FmnI_hq or FmnG_hq and conditions optimized for NrdI were unsuccessful.

The increase in active Mn^III₂-Y^•-NrdF cluster by NrdI recycling likely mimics the assembly process in vivo where a flavodoxin reductase would recycle catalytic amounts of NrdI (5, 23). However, a search of the S. sanguinis genome failed to reveal any candidate homologous to E. coli flavodoxin reductase (FlxR).

Quantitative Characterization of the Association of NrdI_hq with Mn^II₂-NrdF

To assess the affinity of NrdI_hq for Mn^II₂-NrdF, we took advantage of the previous observation in E. coli and B. subtilis systems that NrdI_hq fluorescence is altered by the presence of NrdF (16). Similarly, fluorescence of S. sanguinis NrdI_hq increases 8-fold in the presence of S. sanguinis Mn^II₂-NrdF. Thus, titration studies were carried out as described previously (16), and the analysis reveals a K_d of 2.9 ± 1 μm with 1.5 ± 0.4 NrdI_hq per NrdF. This K_d value is higher than those we previously reported for the NrdI-NrdF interactions in E. coli (K_d < 0.05 μm) and B. subtilis (K_d = 0.6 μm) (16). Given the K_d value and the concentrations of Mn^II₂-NrdF (60 μm) and NrdI_hq (120 μm) used to study cluster assembly described above, >95% of Mn^II₂-NrdF is complexed.

To further explore the similarities/differences among NrdIs from various organisms, an additional experiment was carried out. Previous studies in the E. coli system showed that reduction of NrdI_ox in the presence of apo-NrdF (1 eq) led to formation of an anionic FMN semiquinone (∼30%). In a similar experiment with B. subtilis NrdF, no anionic form was detected (16). We evaluated the FMN form that accumulated upon reduction of S. sanguinis NrdI in the presence of apo-NrdF, and no anionic semiquinone flavin was observed.

Crystallographic Analysis of the S. sanguinis Mn^II₂-NrdF

Because the streptococcal NrdFs are in a phylogenetic group that has not been previously characterized, we determined the structure Mn^II₂-NrdF (2.65 Å resolution, Table 1) to compare with the corresponding structures of the E. coli and B. subtilis NrdFs. As shown in Fig. 4, the Mn^II₂-binding site strongly resembles that of the E. coli Mn^II₂-NrdF structure (15). Each Mn^II ion is six-coordinate with three bridging carboxylates, including the unusual bridging mode for Glu¹⁵⁷ (S. sanguinis numbering), two His ligands, and two coordinated water molecules. The side chain of Asp⁶⁶ hydrogen bonds to Tyr¹⁰⁴, the site of Y^• formation in the active metallo-cofactor, and also resembles the linkage between the corresponding Tyr residue and the metal-binding site in E. coli NrdF. Although the moderate resolution prevents conclusive determination of the orientation of Glu¹⁹¹, the residue is modeled in a μ-η¹,η² binding mode, as observed in E. coli Mn^II₂-NrdF, and the refined model is consistent with this assignment. The structural similarity to E. coli Mn^II₂-NrdF is consistent with similarities observed in the EPR spectra of these proteins.

FIGURE 4.

X-ray structure of S. sanguinis (Ss) Mn^II₂-NrdF (left) in comparison with E. coli (Ec) (middle, PDB accession code 3N37) and B. subtilis (Bs) (right, PDB accession code 4DR0) Mn^II₂-NrdF structures. Mn^II ions (magenta) and water molecules (red) are shown as spheres, and selected amino acid side chains are shown in stick format. An F_o − F_c omit map for the side chain of Glu¹⁵⁷ (chain B) in S. sanguinis Mn^II₂-NrdF is shown in green mesh contoured at 3.5σ.

The unusual configuration of the carboxylate side chains in the E. coli Mn^II₂-NrdF structure opens a solvent-lined channel near Mn2 to the surface of protein (Fig. 5A). The channel is further accommodated by small hydrophobic residues near Mn2 (Ala⁷⁵ and Ile⁹⁴ in E. coli NrdF (53)). B. subtilis Mn^II₂-NrdF, which has a different Mn^II₂ coordination environment and larger Met residues in place of the Ala/Ile pair adjacent to the Mn2 site, does not contain a solvent-occupied channel (53). Because S. sanguinis Mn^II₂-NrdF shares a similar coordination environment with E. coli Mn^II₂-NrdF and retains the hydrophobic residues near Mn2 (Val⁷⁴ and Ile⁹³), it is not surprising that it contains a similar channel (Fig. 5B). The overall cavity shape and volume are nearly identical to the E. coli NrdF channel, and electron density for water molecules is also observed (Fig. 5, C and D). This solvent-lined channel in E. coli Mn^II₂-NrdF is predicted to function as a conduit for the oxidant (O₂^⨪ or HOO(H)) produced by O₂/NrdI_hq to the Mn2 site in Mn^II₂-NrdF. This proposal is based on the structure of the E. coli NrdI-NrdF complex, which revealed a channel extending along the NrdI-NrdF interface, connecting the FMN of NrdI to Mn2 in NrdF. While this manuscript was being revised, the first structures of B. cereus Mn^II₂-NrdF were reported (54) revealing partial occupancy of a bridging μ-1,3 binding mode for the Glu¹⁵⁷ equivalent in this system, contrary to expectation based on the B. subtilis Mn^II₂-NrdF structure (53). These new observations illustrate the capacity for dynamic conformational change in the Mn^II metal-binding site in NrdF. For instance, the B. subtilis Mn^II₂-NrdF coordination environment may transiently switch to an arrangement resembling the E. coli/S. sanguinis Mn^II₂-NrdF structures to facilitate oxidant transport or Mn^II loading.

FIGURE 5.

Solvent channel to the Mn2-binding site in Mn^II₂-NrdF structures. A, E. coli (Ec); B–D, S. sanguinis (Ss). In Ec (A) and Ss (B) Mn^II₂-NrdF structures, the solvent-accessible channel is modeled as a cyan transparent surface. C, 2F_o − F_c electron density map (cyan mesh) contoured at 1.2σ reveals ordered water molecules in the channel in the chain B NrdF monomer. D, zoomed-in view of the modeled waters near the Mn^II₂ site and associated hydrogen-bonding interactions. Selected amino acid side chains and backbone atoms are shown in stick format. Mn^II ions (magenta) and water molecules (cyan) are shown as spheres.

Another interesting conserved feature in the S. sanguinis and E. coli Mn^II₂-NrdF structures involves a constriction in the channel immediately above Mn2. In the E. coli system, the constriction is formed by the side chain of Ser¹⁵⁹ and the backbone carbonyl of the bridging ligand Glu¹⁹², whereas in the S. sanguinis system it involves Thr¹⁵⁸ and Glu¹⁹¹ (Fig. 5, A and B, supplemental Fig. S2). Because oxidant passage through this constriction may be a slow step in metallo-cofactor activation, substitution to a more sterically bulky residue could translate into a slower rate of reaction with the oxidant in the S. sanguinis system.

Crystal Structure of S. sanguinis NrdI_ox

NrdIs have been structurally characterized thus far from several species of Bacillus (50, 51) and E. coli (in complex with Mn^II₂-NrdF) (15). As noted above, streptococcal NrdIs represent a third phylogenetic group for which no structural information exists. Sequence alignments (supplemental Fig. S3) reveal that S. sanguinis NrdI has three distinct insertions. The 1.88 Å resolution crystal structure supports this prediction showing an extension of helix α1 (residues 14–30), insertion of a new helix between the β-strand of β2 and β3 (α1*, residues 40–47), and extension of the loop between β3 and α2 near the FMN cofactor (Fig. 6A). This extended loop is 11 amino acids long (residues 65–76), making it significantly larger than the corresponding loops in E. coli NrdI (eight residues) and the Bacillus NrdIs (three residues).

FIGURE 6.

X-ray structure of S. sanguinis NrdI_ox. A, comparison of the structures of B. subtilis (Bs) NrdI_ox (left, PDB accession code 1RLJ) and S. sanguinis (Ss) NrdI_ox (right). B, close-up view of the fully ordered 70s loop in chain B in S. sanguinis NrdI_ox. C, close-up view of the partially disordered S. sanguinis NrdI_ox chain A 70s loop (disordered residues 67–71 are shown as a heavy gray dashed line) in an alternate conformation. Selected amino acids and the flavin cofactor (yellow) are shown in stick format. Hydrogen bonding interactions are illustrated with thin dashed black lines. The flavin C4a position is marked with an asterisk.

The S. sanguinis NrdI_ox structure contains two copies of the protein in the asymmetric unit and thus provides two independent views of this extended loop's conformation. In one monomer, it is fully ordered and extends directly away from the FMN (Fig. 6B). In the second monomer, it is partially disordered (residues 67–71 are not modeled) and folds in toward the si face of the flavin (Fig. 6C). The fully ordered view of the loop (residues 65–76) shows that it adopts a complex secondary structure composed of an extension of the β-sheet secondary structure from which the loop emanates followed by a series of successive turns. Participation of the loop in an extended β-sheet hydrogen-bonding pattern prevents the 70s loop backbone from interacting directly with the FMN cofactor in the oxidized state. This feature is distinct from what is observed in the E. coli NrdI_ox structure in which the corresponding loop is flexible and glycine-rich, allowing for hydrogen bonding interactions with the N5 of the FMN via a backbone amide NH (15). The short loop (XFG) in the Bacillus NrdIs is similar to S. sanguinis NrdI_ox in that neither can form this interaction. The Bacillus and E. coli NrdIs have also been structurally characterized in the sq- or hq-reduced states (15, 50, 51). All of these structures reveal a backbone amide flip that positions a carbonyl group near the N5 position to interact with the now protonated form of the cofactor.

To understand whether a similar conformational change occurs upon reduction of S. sanguinis NrdI, diffraction datasets were collected on dithionite-soaked NrdI_ox crystals. This treatment reduced the FMN to the sq or hq state, based on observation of color change in the crystals, but the resulting electron density maps did not reveal any significant conformational changes in the 70s loop backbone. The protonated FMN cofactor (FMNH^• or FMNH⁻) interacts instead with a water molecule (not observed in the NrdI_ox structure). The observed outcome in the dithionite-soaked S. sanguinis NrdI crystals could be the result of crystal packing interactions that preclude conformational changes in the loop or it could indicate that the 70s loop backbone cannot, in any oxidation state, bind directly to the FMN N5 position due to the β-sheet hydrogen bonding pattern in the loop near the FMN (Fig. 6B).

The S. sanguinis NrdI 70s loop is also unusual in that it contains many more bulky and negatively charged residues than the corresponding regions in other NrdIs. These residues could affect the electrostatic properties of the flavin and its accessibility to O₂. However, evaluation of the electrostatic environment in NrdI near the FMN isoalloxazine ring shows that the interior of the FMN pocket remains positively charged, similar to what is observed in E. coli and the Bacillus NrdIs (Fig. 7). The negatively charged residues in the S. sanguinis NrdI 70s loop instead generate a strong negative patch on the exterior of the protein. We propose that this patch may be involved in interaction with a flavin reductase required to recycle NrdI_ox in vivo.

FIGURE 7.

Electrostatic surface potential diagram of the S. sanguinis NrdI_ox (chain B) 70s loop and flavin binding pocket is shown contoured at −10 k_BT (red) and +10 k_BT (blue). The FMN cofactor is shown in stick format.

Physiological Reductant for NrdE

The reduction of NDPs to dNDPs is accompanied by oxidation of two active site cysteines in NrdE to a disulfide (Fig. 1) (2, 55). A number of artificial and endogenous systems are capable of mediating this re-reduction step, including dithiothreitol (DTT),Trx/TrxR/NADPH, Grx/GSH/GR/NADPH, and NrdH/TrxR/NADPH, where Trx is thioredoxin, Grx is glutaredoxin, and GR is glutaredoxin reductase (6, 11, 20, 21, 56). The observation that nrdH is colocalized in the same operon with nrdE and nrdF in S. sanguinis and many other organisms makes this protein the most reasonable candidate for the endogenous reductant. Our initial attempts to isolate untagged NrdH were unsuccessful due to its low solubility. To overcome this problem, NrdH was fused to a His₆-SUMO tag and then isolated by nickel-affinity chromatography. Subsequent removal of the tag with SUMO protease gave soluble NrdH, which has been used with TrxR to assay RNR. Two candidate genes (trxR1 and trxR2) for thioredoxin reductases were identified, and the corresponding proteins, TrxR1 and TrxR2, were overexpressed and purified to homogeneity by nickel-affinity chromatography. To ensure that the isolated TrxRs were fully loaded with cofactor, FAD was added to crude cell lysates prior to purification (6, 57). This protocol gave homogeneous FAD-bound TrxR1 and TrxR2 with the ratio A₂₇₂/A₄₅₅ of 6.0 and 6.4, respectively. Complete cofactor loading was further confirmed by anion exchange chromatography.

The turnover number for each TrxR was measured using NADPH, NrdH, and the DTNB assay (45). The K_m value of TrxR1 for NrdH was 0.09 μm and k_cat = 3.5 s⁻¹ giving k_cat/K_m of 4.03 × 10⁷ m⁻¹ s⁻¹. TrxR2, however, showed no activity under the same conditions. Thus, the results suggest that TrxR1 is the reductant for NrdH in vivo.

Activity of Fe^III₂-Y^• and Mn^III₂-Y^• NrdFs Using DTT, NrdH/DTT, and NrdH/TrxR1/NADPH

The activity of the class Ib RNRs has been predominantly reported for the Fe-loaded NrdFs (14, 21, 58, 59), as the role of NrdI in generation of active Mn-loaded RNRs was not elucidated until 2010. Although a number of recent studies have reported activities for Mn^III₂-Y^• NrdFs, in most cases the proteins contained substoichiometric manganese loading and Y^• and/or the endogenous reductant was not used (4–6, 8). As a starting point to identify the metallo-cofactor required for S. sanguinis class Ib in cultures and in an animal model for S. sanguinis-mediated endocarditis, we have measured the activity of pure reconstituted Mn^III₂-Y^• and Fe^III₂-Y^• with DTT, DTT/NrdH, and the endogenous reductants NrdH and TrxR1 (Fig. 1). Some representative results of optimization of the concentrations of NrdE, NrdF, and variable reductants are shown in Fig. 8, A–C.

FIGURE 8.

SA of Mn^III₂-Y^•-NrdF (0.9 Y^•/β₂). A, SA of NrdF in the presence of variable amounts of NrdE using DTT (red) or NrdH/DTT (black) as a reductant. B, SA measured with variable [NrdH], 70 nm [NrdE], [NrdF], 0.1 mm [dATP], and 20 mm [DTT]; C, K_m value for the interaction between NrdF and NrdE using a 1:1 ratio of subunits, 0.1 mm [dATP], 10 μm [NrdH], and 20 mm [DTT]; D, SA measured in the presence of variable dATP concentrations, 70 nm [NrdF], 0.14 μm [NrdE], 10 μm [NrdH], and 20 mm [DTT]. NrdE, specific activity of 3,000 units/mg, was used in all experiments. All plots were fit to Michaelis-Menten equation using IgorPro.

The K_d value for NrdE-NrdF interactions in the class Ib RNRs still remains largely unknown. However, recent studies on B. subtilis class Ib RNR demonstrated that the active form is a 1:1 complex of subunits (5, 47). Thus, our initial studies focused on establishing the ratio of NrdE to NrdF for maximum activity using DTT and NrdH/DTT as reductant. First, Mn^III₂-Y^•-NrdF (0.9 Y^•/β₂) at 0.2 μm was assayed with increasing concentrations of NrdE (0.4–4 μm) and DTT in excess. Under these conditions, maximum activity is observed at 5 eq of NrdE (Fig. 8A, inset). When the assay was carried out with Mn^III₂-Y^•-NrdF (0.07 μm) using NrdH as the reductant, the SA of Mn^III₂-Y^• was 20-fold higher relative to DTT and only 1 to 2 eq of NrdE were required for maximum activity (Fig. 8A). Differences in the NrdE/NrdF ratios required for maximal activity with DTT and NrdH could in part reflect the higher efficiency of NrdH in recycling NrdE, which results in higher concentrations of reduced NrdE available for each turnover. In almost all subsequent assays, a 1:2 ratio of NrdF to NrdE was used.

The effect of NrdH on RNR activity was also examined by maintaining a 1:1 ratio of NrdF/NrdE while increasing the concentration of reduced NrdH. The apparent K_m value for NrdH is 0.4 μm (Fig. 8B). Studies of the DTT requirement for NrdH cycling revealed that levels as low as 1 mm were sufficient for optimal activity. Thus with 0.07 μm Mn^III₂-Y^•, 10 μm NrdH, and 20 mm DTT, a specific activity of 5,700 units/mg was observed. Although DTT is routinely used as a reductant to assay RNR directly and, as just noted, can recycle oxidized NrdH, the endogenous reductant for NrdH is TrxR1. Thus, DTT was replaced with 0.5 μm TrxR1 and 1 mm NADPH and a specific activity of 5,000 units/mg was measured (Table 2). The observed turnover numbers with NrdH/DTT and NrdH/TrxR1/NADPH are very similar and considerably higher than the specific activity of Mn^III₂-Y^•-NrdF reported for other organisms with their endogenous reductants (6, 8). One additional variable, the concentration of 1:1 ratio NrdE and NrdF, was examined. The results, shown in Fig. 8C, reveal a V_max of 6,000 units/mg and a K_m of 6.4 nm.

TABLE 2.

Activity of Mn^III₂-Y^•-NrdF and Fe^III₂-Y^•-NrdF reconstituted in pure proteins with different reductants

Metal cofactor	Y•/β2	SA, units/mg			MeIII/β2
		DTT	NrdH, DTT	NrdH, TrxR, NADPH
Mn	0.9	260 ± 30	5,700 ± 460	5,000 ± 175	3.7 ± 0.2
Fe	1.2	170 ± 40	1,500 ± 360	1,500 ± 100	3 ± 0.5

The SA of Fe^III₂-Y^•-NrdF (1.2 Y^•/β₂) was also measured using the conditions optimized for the Mn^III₂-Y^• and gave an activity of 170 units/mg with DTT, 1,500 units/mg with NrdH/DTT, and 1,500 units/mg with NrdH/TrxR1/NADPH. A comparison of the Mn- and Fe-loaded NrdF activities indicates that the former has a turnover number that is 3.5-fold higher than the Fe-loaded enzyme, establishing that Mn-loaded NrdF is a better catalyst.

In the assays described in Fig. 8, CDP was used as a substrate and dATP (100 μm) as an allosteric effector. Our recent studies of B. subtilis class Ib RNR showed that dATP inhibits RNR activity at concentrations >5 μm (47). This result was surprising because class Ib RNRs do not have an ATP cone or activity domain, the typical binding site for dATP that facilitates class Ia RNR inhibition (52, 60, 61). To determine whether the S. sanguinis class Ib RNR is inhibited by dATP, the activity of Mn^III₂-Y^•-NrdF was measured in the presence of increasing dATP concentrations (Fig. 8D). The apparent K_m is 2.4 μm, and no inhibition was observed even at 1 mm dATP.

DISCUSSION

Since our discovery that NrdI plays an essential role in Mn^III₂-Y^• formation, many reports have appeared describing the activity and properties of manganese-loaded class Ib RNRs (4–8, 22). Although the identification of the endogenous reductant has been reported in some of the studies, in most cases the activity of the class Ib RNR remained very low even with the endogenous reductant (6, 8). We have chosen to examine the importance of manganese- versus iron-loaded cofactor in S. sanguinis class Ib RNR because this organism causes infective endocarditis, and deletion of a manganese transporter results in loss of virulence (25). In addition, its NrdI and NrdF proteins belong to a group phylogenetically distinct from the E. coli, C. ammoniagenes, and B. subtilis class Ib enzymes, the only reported manganese-RNRs at the time we began this work.

Studies of the class Ib RNR have been hampered by the poor efficiency of Mn^III₂-Y^• cluster assembly and consequently low catalytic activity of the enzyme. Our recent work on the B. subtilis class Ib RNR revealed that chromatographic removal of apo- and substoichiometrically metallated NrdF and identification and use of the endogenous reductant (Trx/TrxR) increased the activity of Mn^III₂-Y^•-NrdF to 1,475 units/mg, 18 times that measured for RNR isolated directly from B. subtilis. Furthermore, the activity of the Mn^III₂-Y^•-NrdF was 12 times that observed for Fe^III₂-Y^• (125 units/mg) (47). In a similar study of the B. anthracis class Ib RNR, using the endogenous reductant Trx/DTT, the activity of the Mn^III₂-Y^• was 10 times higher than the Fe^III₂-Y^•. However, the overall activity of Mn^III₂-Y^•-NrdF was only 65 units/mg, possibly associated with the inefficiency of manganese cluster assembly (6). With S. sanguinis RNR, the activity of 6,000 units/mg is very high, but the activity difference between the manganese- and iron-loaded NrdFs is only 3.5-fold using the endogenous reductant, NrdH/TrxR1/NADPH. This result is distinct from all other systems studied to date and raises an important question of which metal is used as a cofactor in vivo and how the organism's environment might influence the enzyme's metallation state.

Class Ib RNRs are found in actinobacteria, firmicutes, and α- and γ-proteobacteria, and recent studies have used bioinformatic analysis of the genomes of interest to identify candidate genes for the endogenous reductant(s) (6, 47). In some organisms, nrdH is found in an operon with nrdE and nrdF, whereas in the Bacillus genus, the nrdH is in a distant location (12). Recent studies of B. anthracis class Ib RNR identified several candidates for its endogenous reductant system as follows: two Trxs, three putative NrdHs, and three TrxRs. Biochemical analysis of the reductant's efficiency (k_cat/K_m) in recycling NrdE, accompanied by Western blot analysis of concentrations of the most interesting reductants, led to the conclusion that both Trx1 and NrdH could function in this capacity but that the former is the most likely candidate in vivo (6). Importantly, the reported V_max for the B. anthracis Mn^III₂-Y^• (45 to 65 units/mg) was 100-fold lower than our S. sanguinis turnover number and independent of the reductants Trx1/DTT, Trx1/TrxR1, NrdH/DTT, and NrdH/TrxR1. Finally, Trx1 had no stimulatory effect at all on the turnover number of Fe^III₂-Y^•, and therefore it was concluded that the manganese-loaded NrdF was the likely form of the class Ib RNR in vivo. Our studies of the B. subtilis class Ib RNR also support this hypothesis by showing that thioredoxins TrxA and YosR (NrdH-like) give a 10- and 5-fold stimulation of Mn^III₂-Y^• activity, respectively, relative to DTT, but they stimulate Fe^III₂-Y^• activity only 2-fold. In contrast, S. sanguinis NrdH significantly increased activity of both Mn^III₂-Y^• and Fe^III₂-Y^• 20-fold and 9-fold, respectively, relative to DTT.

Recently, we have measured an K_m of 25 nm for the B. subtilis class Ib NrdE-NrdF (αβ) subunit interactions, which is 4–10-fold lower than the interactions in the E. coli class Ia RNR (K_d of 0.06 to 0.2 μm (62)). These results suggested that class Ib RNR can be assayed using a 1:1 ratio of subunits (47), instead of with an excess of one subunit over the other to ensure complete αβ complex formation (29). Our studies of S. sanguinis NrdF-NrdE, conducted using a 1:1 ratio of subunits in the concentration range from 0.001 to 0.1 μm (Fig. 8C), gave a K_m of 6.4 nm, similar to the B. subtilis class Ib RNR. The NrdE-NrdF interaction presents an opportunity to crystallize the active αβ complex, and this work is in progress.

The interaction of S. sanguinis NrdF with its NrdI is also of interest because they belong to a third phylogenetic subgroup that remained uncharacterized until our studies (24, 28, 51). This subclassification based on bioinformatics is supported by the measured K_d of 2.9 μm for S. sanguinis NrdI-NrdF interactions, distinct from tighter interactions measured for E. coli (<0.05 μm) and B. subtilis (0.6 μm) systems (16). Although our crystallization efforts have not yielded a structure of the S. sanguinis NrdF-NrdI complex, crystallization of the individual proteins has been successful. As suggested by sequence alignments (supplemental Fig. S2), the coordination environment of the S. sanguinis Mn^II₂-NrdF is much more similar to that of E. coli NrdF than the B. subtilis protein. The three distinguishing features between the E. coli and B. subtilis NrdFs are the H-bonding interactions between the Tyr residue to be oxidized in the active cofactor and the aspartate residue coordinated to Mn1, the presence of a H₂O molecule coordinated to Mn1, and the unusual bridging coordination of Glu¹⁵⁸ to Mn1 and Mn2 (15, 53). Using all three criteria, S. sanguinis Mn^II₂-NrdF is similar to E. coli Mn^II₂-NrdF (Fig. 4). Another distinguishing feature between E. coli and B. subtilis NrdFs is the presence of a water-lined channel linking Mn2 to the FMN in NrdI containing a constriction created by Ser¹⁵⁹ (Fig. 5A). The channel has been proposed to provide a pathway for the oxidant from the flavin in NrdI to the metal-binding site in NrdF. The crystal structure of S. sanguinis Mn^II₂-NrdF reveals the presence of a similar channel with a Thr¹⁵⁸ counterpart to Ser¹⁵⁹ at the constriction (Fig. 5B). The substitution for a bulkier side chain at this site may provide a more stringent selectivity filter for the oxidant. This property may be particularly critical in streptococci because these organisms accumulate high concentrations of H₂O₂ (63, 64), and the observed constriction may restrict NrdF Mn^II₂ site access by this molecule. In addition, the presence of this constriction might explain why Mn^II₂-NrdF cannot be activated directly with H₂O₂ itself (4, 14).

The x-ray structure of S. sanguinis NrdI confirms predictions about the unique features of the streptococcal class Ib proteins. The longer sequence of S. sanguinis NrdI translates into an extended helix α1, an additional helix α1*, and an extended 70s loop when compared with structures of E. coli and B. subtilis NrdIs (Fig. 6A). These extensions could be involved in interaction with another protein, such as a NrdI reductase. A crystal structure of E. coli flavodoxin reductase (FlxR) (65) was used to construct a docking model with S. sanguinis NrdI (models generated with ClusPro (66)). As shown in Fig. 9A, a model was produced that places the long and bulky 70s loop and inserted helix α1* of NrdI in close contact with FlxR near its FAD cofactor. A similar model for interactions between FlxR and flavodoxin suggested that flavodoxin binds in a bowl-shaped pocket close to FAD with positively charged residues of FlxR positioned to interact with conserved negatively charged residues in the flavodoxin (65). A negative electrostatic surface potential near the FMN is a defining characteristic of flavodoxins, but NrdIs instead use positively charged residues to facilitate reduction of FMNH^• to FMNH⁻ (67, 68). Based on analysis of the S. sanguinis NrdI electrostatic surface potential near its FMN cofactor, the negatively charged 70s loop does not seem to influence the positive electrostatic environment around the FMN and instead is localized at the outer surface of the protein (Fig. 7). Thus, we hypothesize that the negatively charged 70s loop may play a role in facilitating interaction between the positively charged pocket of FlxR (Fig. 9B) and the positively charged patch around FMN in NrdI.

FIGURE 9.

Model of an interaction between E. coli FlxR (PDB accession code 1FDR) and S. sanguinis NrdI. A, docking model shows that the 70s loop and α1* of NrdI may interact with FlxR. FlxR is in green; NrdI is in cyan with the 70s loop and α1* in blue, and the FMN (red) and FAD (magenta) cofactors are shown as sticks. The model was generated using ClusPro (66). B, electrostatic surface potential diagram of FlxR near the modeled interface with NrdI is shown contoured at −15 k_BT (red) and +15 kBT (blue). The FAD cofactor is shown in stick format.

Using the E. coli crystal structure of the NrdF-NrdI complex and sequence alignments (supplemental Fig. S2 and S3) with representative class Ib enzymes from the other subgroups, residues involved in forming the NrdF-NrdI interface were predicted for the B. subtilis and S. sanguinis systems. Interestingly, the NrdF portion of the protein-protein interface is more conserved than the NrdI portion. Therefore, differences in the K_d values measured for E. coli, B. subtilis, and S. sanguinis may be due to variations in the NrdI surface (supplemental Table S3) rather than differences in the NrdF component. Moreover, electrostatic analysis of S. sanguinis NrdF revealed that the region surrounding the predicted NrdI-binding site is negative, which, in combination with the negatively charged 70s loop, could account for the particularly low affinity between these proteins.

An active cluster, Mn^III₂-Y^•-NrdF and Fe^III₂-Y^•-NrdF, can be assembled with 1 Y^•/β₂, and both forms exhibit relatively high activity with the physiological reductant NrdH/TrxR. However, the 3.5-fold difference in activities relative to the 10- and 12-fold differences between the Mn^III₂-Y^• and Fe^III₂-Y^• observed with the B. anthracis and B. subtilis NrdFs, respectively, suggests that this organism might be able to stay active in vivo with either cofactor, with loading dependent on the growth environment. The accompanying paper by Rhodes et al. (30) demonstrates in a rabbit model for infective endocarditis that a strain of S. sanguinis in which nrdI has been deleted does not colonize heart valves, unlike the WT-strain. These studies thus provide the first evidence that a class Ib NrdF requires manganese under conditions in which the organism is pathogenic. Considering that streptococci and other pathogens, including enterococci, staphylococci, and Bacillus sp., contain class Ib RNR as their only aerobic RNR, prevention of Mn^III₂-Y^• formation in the class Ib RNR may be an attractive target for new antimicrobials.