Rifamycin antibiotic resistance by ADP-ribosylation: Structure and diversity of Arr

Abstract

The rifamycin antibiotic rifampin is important for the treatment of tuberculosis and infections caused by multidrug-resistant Staphylococcus aureus. Recent iterations of the rifampin core structure have resulted in new drugs and drug candidates for the treatment of a much broader range of infectious diseases. This expanded use of rifamycin antibiotics has the potential to select for increased resistance. One poorly characterized mechanism of resistance is through Arr enzymes that catalyze ADP-ribosylation of rifamycins. We find that genes encoding predicted Arr enzymes are widely distributed in the genomes of pathogenic and nonpathogenic bacteria. Biochemical analysis of three representative Arr enzymes from environmental and pathogenic bacterial sources shows that these have equally efficient drug resistance capacity in vitro and in vivo. The 3D structure of one of these orthologues from Mycobacterium smegmatis was determined and reveals structural homology with ADP-ribosyltransferases important in eukaryotic biology, including poly(ADP-ribose) polymerases (PARPs) and bacterial toxins, despite no significant amino acid sequence homology with these proteins. This work highlights the extent of the rifamycin resistome in microbial genera with the potential to negatively impact the expanded use of this class of antibiotic.

The successful treatment of infectious diseases is under constant threat as a result of the small number of new antibiotics being brought to the clinic and the relentless pressure of evolving resistance to antibiotics (1). Antibiotic resistance manifests itself through a number of mechanisms, including drug efflux and target alteration; however, it is the exquisite power and specificity of enzymatic drug inactivation that is perhaps the apogee of bacterial response to antibiotics (2). These enzymes use a number of strategies to overcome the toxic properties of antibiotics, including destruction of the antibiotic for example by hydrolysis, as well as drug modification by kinases, acyltransferases, adenylyltransferases, and glycosyltransferases. To this impressive arsenal of antibiotic countermeasures, research by Dabbs and colleagues has added ADP-ribosylation (3–10).

ADP-ribosylation is widely recognized as an important mechanism of protein posttranslational modification with numerous biological outputs ranging from modulation of microbial virulence to influencing eukaryotic cell-cycle progression (11). These bacterial and eukaryotic protein (ADP-ribosyl)transferases (ARTs) share similar substrates (all proteins), catalytic mechanisms, and general overall 3D fold (12, 13). In all cases, the ADP-ribosyl donor is NAD⁺, and enzyme action transfers the ADP-ribose unit to a susceptible amino acid residue on the target protein with loss of nicotinamide.

The 3D structures of several protein ADP-ribosyltransferases have been reported including chicken poly(ADP-ribose) polymerase (PARP)-1 (14), rat ART2.2 (15), mosquitocidal toxin from Bacillus sphaericus (16), diphtheria toxin (17), cholera toxin (18), domain III of Pseudomonas aeruginosa exotoxin A (19), iota-toxin from Clostridium perfringens (20), pertussis toxin (21), C2-toxin (22), and C3-toxin (23) from Clostridium botulinum.

Although well established as a protein modification mechanism of significant importance, small molecule ADP-ribosylation is unknown with one exception: a reported ADP-ribosylation of the antibiotic rifampin that results in resistance in bacterial pathogens (5, 6, 9, 10). Rifampin is a semisynthetic derivative of the natural product rifamycin B derived over 40 years ago from Amycolatopsis mediterranei (Fig. 1) (24). Rifamycins inhibit the β-subunit of bacterial DNA-dependent RNA polymerase (RpoB) by obstructing the exit tunnel for nascent RNA (25). Rifampin is used primarily in the combination drug treatment of infections caused by Mycobacterium tuberculosis and is a front line drug in control of this disease (26). Increasingly, rifamycins also find use for the treatment of drug-resistant Staphylococcus aureus (27).

Fig. 1.

Structures of rifamycin antibiotics of clinical importance. The rifamycin antibiotics consist of an aromatic naphthyl core and a polyketide derived ansa-bridge.

Consistent with the lack of evidence for plasmid-mediated horizontal gene transfer in mycobacteria, rifampin resistance in M. tuberculosis, including extensively drug-resistant tuberculosis, results from point mutations in rpoB that lower affinity for the drug (28). In the opportunistic pathogen Mycobacterium smegmatis, however, rifampin is an ineffective drug because of the presence of a chromosomally encoded rifampin ADP-ribosyltransferase (Arr-ms). Arr-ms is a small enzyme (≈16 kDa) with no sequence similarity to known protein ADP-ribosyltransferases such as ART, PARP, and bacterial toxins (6, 29). Paradoxically, a homologue of Arr, Arr-2 (55% identical with Arr-ms), also is present in several transposons and integrons found in Gram-negative pathogenic bacteria such as P. aeruginosa (30), Escherichia coli (31), Klebsiella pneumoniae (32), and Acinetobacter baumannii (33). This distribution of arr-2 in Gram-negative pathogens is unexpected because rifampin is not used to treat infections caused by these bacteria, and therefore the selective pressure for maintaining this resistance cassette would be predicted to be low unless the patient was receiving rifampin treatment for other infections and as a result this has selected for “bystander” resistance in Gram-negative bacteria.

The rifamycin class of antibiotics has been revisited in recent years as a scaffold worthy of investigation for the development of new drugs (Fig. 1). Rifaximin, a semisynthetic rifamycin, was approved in 2003 for prophylactic use in the treatment of traveler's diarrhea (34). Rifalazil, an analogue with antichlamydial activity (35), is in Phase III trials. This rediscovery of the rifamycin antibiotics continues apace, and new semisynthetic rifamycin derivatives are in various stages of development (e.g., refs. 36–38).

With the growing importance of rifamycin antibiotics in antimicrobial therapy, there is opportunity for the selection of preexisting resistance elements such as Arr-2 and for opportunistic pathogens with chromosomal Arr-like elements and their subsequent mobilization and transfer to human pathogens. Here, we report the characterization of the enzymatic efficacy of several Arr orthologues and their activity with a panel of rifamycins and the 3D structure of Arr-ms from M. smegmatis in complex with rifampin. The structure reveals similarity to protein ADP-ribosyltransferases, despite a lack of amino acid sequence similarity, and provides another example with protein kinases (39) and protein acetyltransferases (40, 41) of the reengineering of important protein modifying enzyme scaffolds familiar to eukaryotic biology in antibiotic resistance.

Results

Diversity and Distribution of arr in the Resistome.

Using the Arr-2 protein sequence as a query, we identified a number of possible Arr orthologues in microbial genome and metagenome databases. All of these similar proteins were predicted to be 15–18 kDa and showed >55% sequence similarity. Sequence alignment revealed a broad distribution of predicted Arr orthologues throughout microbial genera [supporting information (SI) Fig. 5 in SI Appendix]. Many, but not all, of these arr-like genes are either on mobile genetic elements (e.g., arr-2 in Gram-negative pathogens) or are clustered in the genome with gene sequences (e.g., predicted transposase encoding genes) that suggest historic acquisition by horizontal gene transfer (SI Fig. 6 in SI Appendix).

The sequence search identified arr homologues in environmental bacteria, including opportunistic pathogens such as M. smegmatis, Stenotrophomonas maltophilia, and Burkholderia cenocepacia, as well as a number of nonpathogenic bacteria, including the actinomycetes Streptomyces coelicolor and Corynebacterium glutamicum, the anaerobic firmicute Desulfitobacterium halfniense, and the α-proteobacterium Rhodopseudomonas palustris. Furthermore, the number of complete and partial arr-like sequences derived from environmental metagenome studies from marine and terrestrial samples speaks to the expansive distribution of Arr-like proteins in the environment even in the absence of obvious human pressure.

Arr-like genes therefore are widespread in a diversity of bacterial genomes. Homology, however, is not equivalent with orthology. We selected three representative genes for further study of their associated enzymatic activities: arr-2 from P. aeruginosa, which is identical to the gene found on several transposons and integrons isolated from a variety of clinical isolates of Gram-negative pathogens; arr-ms, from the genome of the opportunistic pathogen M. smegmatis; and arr-sc, found in the genome of the benign soil bacterium S. coelicolor.

Characterization of Arr Enzymatic Activity.

Each of the predicted Arr orthologues was successfully expressed and purified from E. coli (SI Fig. 7 in SI Appendix). Using an HPLC-based enzyme assay with mass spectrometric detection, we confirmed that all three enzymes efficiently catalyzed ADP-ribosylation of rifampin in the presence of NAD⁺ as a cosubstrate (Fig. 2). NADH and NADP⁺ were not substrates for the enzymes.

Fig. 2.

Arr-2 catalyzes rifampin ADP-ribosylation. HPLC analysis of rifampin ADP-ribosylation by Arr-2.

Using this HPLC assay, we compared the steady-state kinetic parameters of the enzymes (Table 1). Remarkably, regardless of source, all of the enzymes displayed very similar kinetic behaviors and constants and are equally effective antibiotic resistance enzymes. The k_cat/K_m ratios measure the efficiency of the enzyme to catalyze the reaction at low substrate concentrations; these were essentially identical, varying only 3-fold. This ratio is most important as the antibiotic begins to enter the cell and therefore serves as an indicator of effectiveness as a drug inactivation mechanism (42).

Table 1.

Steady-state kinetic parameters of Arr enzymes

Enzyme	Substrate	Km, mM	KI, mM	kcat, s−1	kcat/Km, s−1·M−1
Arr-ms	Rifampin	1.42 ± 0.38	1.07 ± 0.35	2.84 ± 0.57	2.00 × 103
	NAD+	0.18 ± 0.04			1.58 × 104
Arr-sc	Rifampin	2.80 ± 0.80	0.20 ± 0.07	7.34 ± 1.90	2.62 × 103
	NAD+	0.31 ± 0.11			2.37 × 104
Arr-2	Rifampin	0.74 ± 0.34	0.55 ± 0.30	4.48 ± 1.49	6.05 × 103
	NAD+	0.18 ± 0.05			2.49 × 104

The Arr-2 enzyme efficiently modified rifamycin antibiotics with diverse naphthyl substituents, including the parent natural product rifamycin SV (no substituent) and the semisynthetic drugs rifampin (antituberculosis drug), rifabutin (for treatment of Mycobacterium avium complex), and rifaximin (prophylactic treatment of traveler's diarrhea) (SI Table 4 in SI Appendix; see Fig. 1 for chemical structures). Arr therefore has a broad ability to inactivate both “old” and new rifamycin antibiotics.

The in vivo efficiency of this inactivation was assessed by determination of the minimal inhibitory concentrations (MICs) of rifamycin antibiotics required to arrest cell growth. In the presence of recombinant arr genes in E. coli, MIC values of all of the tested antibiotics were greatly increased, and under conditions where the genes were induced, MICs all increased to ≥512 μg/ml (Table 2). For comparison, MIC of wild-type M. smegmatis expressing arr-ms is 32 μg/ml (27), well above breakpoint concentrations of this drug (≈2 μg/ml). Therefore, Arr is a potent rifamycin resistance element with broad antibiotic substrate specificity in vitro and in vivo.

Table 2.

MICs of rifamycins against E. coli-harboring Arr orthologues

Arr and Plasmid Vector	MIC, μg/ml
	IPTG	Rifabutin	Rifampin	Rifamycin SV	Rifaximin
pET28a(+)	−	8	4	64	8
pET28a(+)	+	8	8	64	16
pET28a(+)-Arr-2	−	>512	512	>512	>512
pET28a(+)-Arr-2	+	>512	>512	>512	>512
pET22b(+)	−	16	8	32	16
pET22b(+)	+	16	8	64	16
pET22b(+)-Arr-ms	−	64	128	512	>512
pET22b(+)-Arr-ms	+	>512	>512	>512	>512
pET15b(+)	−	16	8	32	16
pET15b(+)	+	16	8	32	16
pET15b(+)-Arr-sc	−	64	128	512	>512
pET15b(+)-Arr-sc	+	512	512	>512	512

IPTG, isopropyl β-d-thiogalactopyranoside.

The structure of the ADP-ribosyl-rifampin product was determined by NMR and mass spectrometric analysis of the purified compound. Mass spectra confirmed the predicted molecular mass of the product (SI Table 4 in SI Appendix). Through a series of 1D and 2D NMR experiments, the regiospecificity of modification was determined to be on the hydroxyl linked to C23 in the ansa-bridge (see SI Experimental Procedures in SI Appendix). This result is consistent with the findings of a previous report of Arr-ms activity (5). We further determined the absolute conformation of the ADP-ribosylated product and found that the reaction occurs with inversion of configuration at C1 of the ribose of NAD⁺ (β → α).

Crystal Structure of Rifampin-Bound Arr-ms.

One of the intriguing features of the rifampin-inactivating Arr family of ARTs is their small size (≈16 kDa). Previously characterized protein modifying ARTs are much larger, typically >40 kDa. In addition, the Arr family shares no obvious amino acid sequence conservation with any of the dozens of known or proposed ARTs.

We determined the structure of Arr-ms in complex with rifampin to 1.45 Å (PDB ID code 2HW2). The final model contained no disordered regions and was refined to R and R_free values of 13.4 and 18.5, respectively. SI Fig. 8 in SI Appendix provides an F_o − F_c omit map of bound rifampin. Complete data collection and model refinement statistics are listed in SI Table 9 in SI Appendix.

Arr-ms contains a single domain comprised of two antiparallel β-sheets and two α-helices situated to one side, forming a second layer in the overall αβ fold (Fig. 3). The first β-sheet, encompassing four strands (β1-4-7-2) is arranged almost perpendicular along the edge of the sheet to the second three-stranded β-sheet (β3-6-5). Helix 1 (α1) is situated against one side of the extended β-sheet region, making closer contact with the first four-stranded β-sheet. Together, α1 and α2 form the walls of a deep rifampin-binding cleft. Helix 2 is present at the C terminus of Arr-ms and participates in an overall wrapping of the C-terminal region around the core domain of Arr-ms. Given the limited contact that α2 makes with the rest of the protein, it is likely that this helix would be unstructured in the absence of rifampin. Rifampin makes significant contact not only with α1 and α2 but also with the extended loop region connecting β5 and β6. This β5–β6 loop serves as a lid, partially covering rifampin once positioned into its deep binding cleft. The most remarkable feature of the interaction between rifampin and Arr-ms is that despite burying 620 Ų of surface area, not one H bond or ionic interaction exists between rifampin and a side chain of Arr-ms. Two H bonds are formed with main chain atoms from N86 and K91, and an additional six water-mediated interactions also are present. The remaining direct interactions are entirely hydrophobic in nature. SI Table 10 in SI Appendix gives a complete list of rifampin–Arr-ms interactions. The apparent plasticity of interactions observed between rifampin and Arr-ms suggest that Arr-ms may be able to act on other substrates in addition to rifampin.

Fig. 3.

Structure of rifampin-bound Arr-ms. Orthogonal views of Arr-ms in complex with rifampin (yellow) and the dipeptide glycylglycine (purple).

Comparison of Arr-ms with Other ADP-Ribosyl Transferases.

Representative crystal structures of ART family members from both eukaryotes (PARPs and ARTs), and prokaryotes (ADP-ribosylating toxins) have been determined (reviewed in refs. 12, 13, and 43). ARTs can be classified into two subfamilies based on 3D structure: class I, characterized by PARP-1 and diphtheria toxin structures, and class II, characterized by ART2 and VIP2 toxin structures. Each class builds on a common NAD⁺-binding core of 5 β-strands arranged as two adjoining sheets. Distinguishing class features include the presence of distinct motifs (H-Y-E or R-S-E) and the addition of a variable number of conserved α-helices above and below the core β-sheet structure.

Comparative structural analysis using the program Dali (44) revealed limited but significant similarity with three previously determined structures: PARP catalytic fragment from chicken (Gallus gallus; PDB ID code 1A26), exotoxin A domain III from P. aeruginosa (PDB ID code 1AER), and a putative ART from the archaeon Aeropyrum pernix (1WFX). Only a small amount of sequence (≈80 residues) within these proteins could be aligned with Z scores ranging from 3 to 5 and rmsd values <3 Å. Nevertheless, as shown in Fig. 4, Arr-ms could be structurally aligned with the conserved core region from other ARTs. Comparison of topology (SI Fig. 9 in SI Appendix) further suggested that Arr-ms belongs to the class I subfamily of ARTs. As demonstrated in SI Fig. 9 in SI Appendix, Arr-ms represents a minimal ART unit, containing only the essential elements necessary for NAD⁺ binding and catalysis. For simplicity, we hereafter refer to the Arr-ms labeling of topology in making comparison to other ART family members.

Fig. 4.

Structural alignment of the core NAD⁺ binding cleft conserved among ADP-ribosyl transferase enzymes. The structure of Arr-ms (yellow) in complex with rifampin is shown aligned in stereo with the structurally conserved NAD⁺ binding cleft of domain III of P. aeruginosa exotoxin (green), PARP catalytic fragment (purple), and a probable 2′-RNA phosphotransferase (blue) (PDB ID codes 1AER, 1A26, and 1WFX, respectively). The structure of domain III of P. aeruginosa exotoxin was solved in complex with β-methylene-thiazole-4-carboxamide adenine dinucleotide shown here in light blue to highlight the NAD⁺ binding site.

Class I ARTs contain a highly conserved H-Y-E motif; however, standard sequence alignment algorithms fail to reveal the presence of such a motif in Arr-ms. Using a structure-based sequence alignment (SI Fig. 10 in SI Appendix), we identified two residues (H19 in β1 and Y49 in β3) structurally conserved across Arr and remaining class 1 ARTs. H19 and Y49 make up part of the so-called H-Y-E motif (43) and are predicted to contribute directly to NAD⁺ binding. We mutated both His-19 and Y49 to Ala, and these showed no in vitro activity and reduced MIC levels of rifampin to control levels of susceptibility (Table 3), consistent with a vital role in enzyme activity.

Table 3.

In vitro rifampin susceptibility of Arr-ms mutants

Construct in E. coli	MIC, μg/ml
pET22b(+) (negative control)	16
Arr-ms (positive control)	256
His-19–Ala	4
Tyr-49–Ala	4
Asp-84–Ala	8

Previous studies have shown that only a single residue, the catalytic glutamic acid present on the front edge of β6, is absolutely conserved in all structures of known functional ARTs. This residue is essential for stabilizing the predicted oxocarbenium ion in the transition state (13, 45). Remarkably, this residue is missing from Arr-ms. Instead an aspartic acid residue (Asp-84) located within the substrate-binding loop connecting the less conserved fifth β-strand to β6, appears to fulfill this function in Arr-ms. There is no obvious structural need for this to have occurred; however, positioning the key catalytic residue in the substrate-binding loop would be an effective way of preventing formation of a functional active site before substrate binding. Mutation of Asp-84 to Ala abolished both in vitro ADP-ribosylation and in vivo rifampin resistance as predicted if this residue fulfills the role of the invariant Glu in protein ARTs.

Discussion

ADP-ribosylation of rifampin in M. smegmatis cell-free extract (3, 5) and by purified Arr-2 (this work) occurs on the hydroxyl group at C23 of the antibiotic and with inversion of configuration at C1 of the NAD⁺-derived ribose. The C23 hydroxyl group interacts with the amide backbone of Phe-394 in the crystal structure of rifampin bound to the Thermus aquaticus RNA polymerase (25), providing the molecular basis for Arr-mediated resistance. The substrate specificity of Arr spans old antibiotics such as rifampin and the natural product rifamycin SV as well as new agents such as rifaximin. The location of modification on the ansa-bridge of the antibiotic at position 23 and the tolerance of substitutions on the naphthyl ring indicate that it is very likely that several new rifamycin derivatives being considered for clinical use also will be susceptible to Arr-catalyzed inactivation.

Substrate binding of ARTs is perhaps the least understood aspect of ART function. For class I ARTs, the loop connecting β5 and β6 has been suggested to function in substrate recognition. Our structure of rifampin-bound Arr-ms provides structural evidence to support this claim because the β5–β6 loop of Arr-ms does indeed form the lid to the rifampin-binding pocket. A significant difference between Arr and other ARTs is the presence of the C-terminal helix α2, which serves as a platform for rifampin binding and physically blocks access to the active site by large substrates such as proteins. As noted above, none of the direct contacts mediated by Arr-ms side chains involves a H bond to rifampin. The functional significance of the highly conserved lysine residues (K90 and K91) within the substrate-binding loop is not apparent. These residues may be important for Arr activity directed toward other uncharacterized substrates. The absence of direct interactions of antibiotic with the enzyme, the broad substrate specificity, and the similarity of kinetic specificity constants between enzymes suggests that Arr enzymes likely have other functions in the cell, possibly as small molecule ARTs, and that rifamycin antibiotic resistance is a secondary or fortuitous activity, which would be consistent with unexpected broad distribution of arr in microbial genomes in the absence of obvious antibiotic selection.

The structure of Arr-ms, along with site-directed mutagenesis studies and literature precedent for other ARTs, is consistent with a molecular mechanism where Asp-84 stabilizes a developing oxocarbenium transition state enabling attack of the hydroxyl group at position 23 of the antibiotic to attack C1 of the ribose (Scheme 1). This action would result in inversion of stereochemistry in the ADP-ribosylated product at this position, which we confirmed experimentally. This mechanism predicts that transition state inhibitors of ARTs that take advantage of the predicted structure and electronic configuration of the transition state, e.g., ref. 46, could find use as inhibitors of Arr and perhaps overcome resistance in vivo.

Scheme 1.

Proposed mechanism of Arr-catalyzed rifampin inactivation.

Recently, new rifamycins with carbamate derivatization at position 25 have been reported that maintain antibiotic activity in the presence of Arr (38). The crystal structure of rifampin bound to Arr-ms provides the logic for evasion of inactivation by these new compounds because the bulky carbamate derivatives reported would be unable to bind productively to the enzyme, thereby avoiding modification. Additional modifications of the ansa-bridge at adjacent positions should prove equally effective in avoiding Arr-mediated resistance.

Significance.

The Arr family of proteins is widely distributed in microbial communities. Although this mechanism is not yet highly prevalent in pathogenic bacteria, the growing number of new rifamycin antibiotics either already in the clinic or in development for the treatment of nontuberculosis infections provides the perfect opportunity to select for Arr-mediated resistance. This study of the distribution, activity, structure, and mechanism of Arr provides an early warning that enzymatic resistance to rifamycin antibiotics is prevalent and mobilizable. The 3D structure and function studies presented here provide the molecular rationale for strategies to overcome Arr such as through alteration of the ansa-bridge of the antibiotic (e.g., ref. 38) and the development of Arr inhibitors that have the potential to overcome this threat to the next generations of rifamycin antibiotics. Furthermore, this work provides another example of the exploitation of important protein scaffolds by evolution resulting in antibiotic resistance. As we begin to more fully appreciate the depth of the resistome, it is likely that further examples of this replication of protein structure and mechanism will emerge.

Methods

Overexpression and Purification of Arr Enzymes.

Arr enzymes were expressed as hexa-His fusions and purified by Ni-affinity and ion exchange chromatography (see SI Experimental Procedures in SI Appendix for further detail).

Analytical Procedures.

Reverse-phase (RP)-HPLC and liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) were used to confirm the presence of ADP-ribosyl rifampin before assay development. RP-HPLC was performed with a C₁₈ column (3 μm, 120 Å, 4.6 × 150 mm). The activity of rifampin ADP-ribosyl transferase enzymes Arr-ms, Arr-sc, and Arr-2 was analyzed by using a dual solvent system consisting of 0.05% trifluoroacetic acid (TFA) in water (vol/vol) and 0.05% TFA in acetonitrile (vol/vol). LC/ESI-MS data were obtained by using an Agilent 1100 Series HPLC system and an Applied Biosystems QTRAP LC/MS/MS system. For LC/ESI-MS analysis, formic acid was used as a counter ion instead of TFA.

Assay Development and Kinetics.

Progress curves were generated for each of the Arr enzymes at three different substrate concentrations and used to determine the linear initial rate range of the reaction. Reactions were prepared in 100 μl of 50 mM Hepes, pH 7.5, 1 nmol of Arr, and 0.1 mM, 0.5 mM, or 1.0 mM NAD⁺ or rifampin and stopped by the addition of methanol to 50% (vol/vol). Product formation was analyzed by RP-HPLC as described above. A calculated conversion factor based on the area under the curve (AUC) of a fixed concentration of rifampin at 340 nm was used to convert the product AUC into units of molarity.

Steady-state kinetic parameters for rifampin and NAD⁺ were determined for the Arr-ms and Arr-2 enzymes by varying substrate at six concentrations from 0.05 to 2.0 mM, and for the Arr-sc enzyme by varying substrate at five concentrations from 0.05 to 1.0 mM. Reactions were prepared in 100 μl of 50 mM Hepes, pH 7.5, 1 nmol of Arr, 0.5 mM NAD⁺, and varied rifampin concentrations to determine steady-state kinetics for rifampin, and in 100 μl of 50 mM Hepes, pH 7.5, 1 nmol of protein, 0.5 mM rifampin, and varied concentrations of NAD⁺ to determine steady-state kinetics for NAD⁺. All reactions were set up in duplicate. Reaction mixes were incubated for 5 min, stopped by the addition of methanol to 50% (vol/vol), and analyzed by HPLC for product formation as described above.

Arr Structure Determination.

Arr-ms was crystallized in the presence of rifampin by using hanging drop-vapor diffusion. Single-wavelength anomalous dispersion (SAD) phasing was performed with selenomethionine-derivatized Arr (see SI Experimental Procedures in SI Appendix for further detail of crystal growth, data collection, and model refinement).

Site-Directed Mutagenesis of Arr-ms.

Site-specific mutations H19A, Y49A, and D84A were introduced into the arr-ms gene by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Oligonucleotide primers are listed in SI Table 11 in SI Appendix. The pET22b (+) vectors carrying the amplified genes for the arr mutants were used to transform E. coli BL21 (DE3) cells. Mutants were confirmed by DNA sequencing.

MIC Determinations.

The MICs were determined by broth dilution in Mueller–Hinton broth (MHB). Colonies of E. coli BL21 (DE3) cells were picked from fresh overnight plates and mixed with 0.85% saline to an OD₆₂₅ of 0.08–0.1. Aliquots of 100 μl of the saline solution were mixed into 18.9 ml of MHB. After this, 95 μl of the mixture was added to each well of a sterile 96-well microtiter plate containing serial dilutions of rifampin at concentrations ranging from 512 to 0 μg/ml. Each well contained 5 μl of rifampin dilution. The plate was incubated overnight at 37°C and assessed for bacterial growth.