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Acta Crystallographica Section F: Structural Biology and Crystallization Communications logoLink to Acta Crystallographica Section F: Structural Biology and Crystallization Communications
. 2013 Feb 27;69(Pt 3):306–312. doi: 10.1107/S1744309113003060

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus

Rachel A North a, Sarah A Kessans a, Sarah C Atkinson b, Hironori Suzuki a, Andrew J A Watson c, Benjamin R Burgess d, Lauren M Angley d, André O Hudson e, Arvind Varsani a,f, Michael D W Griffin d, Antony J Fairbanks c, Renwick C J Dobson a,d,*
PMCID: PMC3606580  PMID: 23519810

N-Acetylneuraminate lyase, an enzyme involved in the bacterial uptake and metabolism of sialic acid, is a promising target for antibiotic development against pathogenic bacteria. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylneuraminate lyase from methicillin-resistant S. aureus to 1.70 Å resolution are reported.

Keywords: antibiotic resistance, N-acetylneuraminate lyase, NAL, sialic acid metabolism, Staphylococcus aureus, MRSA

Abstract

The enzyme N-acetylneuraminate lyase (EC 4.1.3.3) is involved in the metabolism of sialic acids. Specifically, the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-d-mannosamine and pyruvate. Sialic acids comprise a large family of nine-carbon amino sugars, all of which are derived from the parent compound N-acetylneuraminic acid. In recent years, N-acetylneuraminate lyase has received considerable attention from both mechanistic and structural viewpoints and has been recognized as a potential antimicrobial drug target. The N-acetylneuraminate lyase gene was cloned from methicillin-resistant Staphylococcus aureus genomic DNA, and recombinant protein was expressed and purified from Escherichia coli BL21 (DE3). The enzyme crystallized in a number of crystal forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.70 Å resolution in space group P21. Molecular replacement indicates the presence of eight monomers per asymmetric unit. Understanding the structural biology of N-acetylneuraminate lyase in pathogenic bacteria, such as methicillin-resistant S. aureus, will provide insights for the development of future antimicrobials.

1. Introduction  

Staphylococcus aureus is a Gram-positive bacterial pathogen frequently found on the skin and in the nasopharynx of humans (Furuya & Lowy, 2006 ▶). Humans appear to be a natural reservoir for S. aureus; longitudinal studies have demonstrated that nasal colon­ization by S. aureus is persistent in approximately 20% (range 12–30%) of individuals, intermittent in around 30% (range 16–70%) of individuals and non-existent for about 50% (range 16–69%) of individuals (Eriksen et al., 1995 ▶; Hu et al., 1995 ▶; Kluytmans et al., 1997 ▶; Nouwen et al., 2004 ▶; Wertheim et al., 2005 ▶). S. aureus has the potential to become extremely infectious, triggering a wide range of clinical diseases including skin and soft tissue infections, bloodstream infections, pneumonia, osteomyelitis and infective endocarditis (Furuya & Lowy, 2006 ▶). Higher rates of colonization and infection are often observed in patients with type I diabetes (Tuazon et al., 1975 ▶), human immunodeficiency virus (Weinke et al., 1992 ▶), intravenous drug users (Tuazon & Sheagren, 1974 ▶) and those receiving haemodialysis (Yu et al., 1986 ▶).

Since Alexander Fleming’s first observation in 1928 that penicillin had antibiotic properties against staphylococci (Hare, 1982 ▶), S. aureus has continuously evolved resistance mechanisms against novel antimicrobials (Furuya & Lowy, 2006 ▶). In 1959, the first semi-synthetic β-lactam antibiotic named methicillin was introduced. Despite initial successes, by 1961 the first methicillin-resistant S. aureus strains were emerging, which were resistant to virtually all β-lactam antibiotics and their derivatives (Jevons, 1961 ▶). As a result of this selective antibiotic pressure, there has been a dramatic increase in the number of methicillin-resistant S. aureus infections worldwide (Boyce et al., 2005 ▶; Chambers, 1988 ▶; McDonald et al., 1981 ▶; Panlilio et al., 1992 ▶; Speller et al., 1997 ▶). Previously associated with exposure in healthcare settings, this superbug now accounts for an increasing number of superadaptable community-acquired methicillin-resistant S. aureus infections (David & Daum, 2010 ▶). The ever-increasing prevalence of methicillin-resistant S. aureus and the continual progression of antibiotic resistance highlight the need to investigate novel drug targets against this pathogen.

Sialic acids comprise a large family of nine-carbon amino sugars, all of which are derived from the parent compound N-acetylneuraminic acid (Almagro-Moreno & Boyd, 2009a ▶; Vimr et al., 2004 ▶). Interestingly, some pathogenic bacteria can catabolize sialic acid into carbon and nitrogen precursors by scavenging it from their surrounding environment (Almagro-Moreno & Boyd, 2009a ▶; Vimr et al., 2004 ▶). This sequestration and subsequent catabolism of sialic acid requires a well characterized cluster of genes confined to predominantly commensal and pathogenic species of bacteria, known as the ‘Nan–Nag cluster’ (Almagro-Moreno & Boyd, 2009a ▶). The Nan–Nag cluster of genes encodes enzymes that scavenge sialic acid from the host, transport it into the bacterial cell and catabolize it into fructose-6-phosphate (Fig. 1 ▶). This pathway has been well documented in several bacterial pathogens such as Escherichia coli, Haemophilus influenzae, S. aureus, Vibrio cholerae, Vibrio vulnificus and Yersinia pestis (Almagro-Moreno & Boyd, 2009a ▶,b ▶; Chang et al., 2004 ▶; Jeong et al., 2009 ▶). All of these pathogens colonize heavily sialylated mucous-rich niches, such as the human respiratory tract and gut, suggesting that the ability to utilize sialic acid as a ubiquitous carbon and nitrogen source may be important for colonization and persistence (Almagro-Moreno & Boyd, 2009b ▶). Importantly, a correlation between the Nan–Nag cluster of genes, pathogen colonization and pathogen persistence has been proven in mouse models for E. coli (Chang et al., 2004 ▶), V. cholerae (Almagro-Moreno & Boyd, 2009b ▶) and V. vulnificus (Jeong et al., 2009 ▶), making the pathway a viable and admirable target for antimicrobial drug design.

Figure 1.

Figure 1

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Sialic acid metabolism. NanH, neuraminidase; NanT, transporter; NanA, lyase; NanK, kinase; NanE, epimerase; NagA, deacetylase; NagB, deaminase.

N-Acetylneuraminate lyase (also known as sialic acid aldolase) catalyzes the retro-aldol cleavage of N-acetylneuraminic acid (sialic acid, Neu5Ac) to form N-acetyl-d-mannosamine (ManNAc) and pyruvate (Fig. 2 ▶) via a Schiff base intermediate (Barbosa et al., 2000 ▶; Izard et al., 1994 ▶). The enzyme has recently been recognized as a potential antimicrobial drug target (Severi et al., 2007 ▶; von Itzstein, 2007 ▶). The N-acetylneuraminate lyase subfamily of (βα)8-barrel enzymes all share a common structural framework, but catalyze different reactions on separate biochemical pathways (Lawrence et al., 1997 ▶). Members of this subfamily identified thus far include the archetype N-acetylneuraminate lyase (Izard et al., 1994 ▶), dihydro­dipicolinate synthase (Kefala et al., 2008 ▶; Burgess, Dobson, Bailey et al., 2008 ▶; Dobson et al., 2005 ▶; Evans, 2006 ▶; Mirwaldt et al., 1995 ▶), d-5-keto-4-deoxyglucarate dehydratase (Jeffcoat et al., 1969a ▶,b ▶), 2-keto-3-deoxygluconate aldolase (Buchanan et al., 1999 ▶), trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (Eaton, 1994 ▶) and trans-2′-carboxybenzalpyruvate hydratase-aldolase (Iwabuchi & Harayama, 1998 ▶).

Figure 2.

Figure 2

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The reaction catalyzed by N-acetylneuraminate lyase.

On the basis of X-ray structural analysis, the E. coli N-acetyl­neuraminate lyase has been shown to be a homotetramer, with each subunit consisting of a (β/α)8-barrel domain followed by an extension of three α-helices at the carboxy terminus (Izard et al., 1994 ▶). Members of the N-acetylneuraminate lyase subfamily are all involved in the formation of a Schiff base between a strictly conserved lysine residue and the C2 carbon of an α-keto acid moiety on respective substrates; further X-ray structural analysis of covalent complexes of E. coli N-acetylneuraminate lyase with pyruvate and the substrate analogue 3-hydroxypyruvate provides evidence for this notion (Lawrence et al., 1997 ▶). The three-dimensional structures of N-acetyl­neuraminate lyase from H. influenzae in its native form and in complex with three substrate analogues (sialic acid alditol, 4-oxo-sialic acid and 4-deoxy-sialic acid) have also been determined by X-ray crystallography, revealing the mode of substrate binding within the N-acetylneuraminate lyase active site (Barbosa et al., 2000 ▶).

Understanding the structural biology of N-acetylneuraminate lyase in methicillin-resistant S. aureus and other pathogenic species will provide insights necessary for the development of novel antimicrobial agent(s) that target the enzyme in question. Here we present the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylneuraminate lyase from methicillin-resistant S. aureus subspecies aureus MRSA252.

2. Materials and methods  

2.1. Cloning, expression and purification of N-acetylneuraminate lyase  

The nanA structural gene encoding N-acetylneuraminate lyase and flanking nucleotide sequence were amplified by PCR using the primer pair SA_nanA-1F (5′-CGA TTG CGA TGT AAT ACA CC-3′) and SA_nanA-1R (5′-CAT TGT AAA GGG GAT GTT GC-3′) from genomic DNA derived from methicillin-resistant S. aureus subspecies aureus MRSA252. The amplified product was cloned into pCR-Blunt II-TOPO (Invitrogen) to produce a recombinant construct named pRN01. Following sequence verification, the primer pair SA_nanA_ORF-F (5′-CAT ATG AAC AAA GAT TTA AAA GG-3′) and SA_nanA_ORF-R (5′-CAT ATG CTA TAA ATC GTA TTT TG-3′) facilitated PCR amplification and cloning of the nanA open reading frame from pRN01 into the NdeI restriction site of the pET11a (Invitrogen, USA) expression vector to produce pRN02.

The recombinant expression vector, pRN02, was transformed into E. coli BL21 (DE3) cells which were cultured in Luria Broth media (with 50 µg ml−1 ampicillin). Cultures were grown for approximately 5 h at 310 K and 200 rev min−1, until an OD600 of 0.6 was reached. Expression of N-acetylneuraminate lyase was induced by the addition of isopropyl β-d-1-thiogalactopyranoside to a final concentration of 1 mM, followed by further incubation at 310 K and 200 rev min−1 overnight. Cells were harvested by centrifugation (8000 rev min−1, 277 K, 5 min), resuspended in buffer consisting of 20 mM Tris–HCl pH 8.0 and lysed by sonication using a Vibra-cell VC 750 (SONICS). Cell debris was pelleted by centrifugation (10 000 rev min−1, 277 K, 10 min).

Anion-exchange chromatography was performed using a Q Sepharose column (GE Healthcare), previously washed with three column volumes of 20 mM Tris–HCl pH 8.0. Cell lysate was loaded onto the Q Sepharose column and bound N-acetylneuraminate lyase was eluted using an increasing gradient to 20 mM Tris–HCl pH 8.0, 1 M NaCl. Hydrophobic interaction chromatography was implemented as the second purification step; 1 M ammonium sulfate was added to pooled fractions containing N-acetylneuraminate lyase from the anion-exchange step and then applied to a Phenyl Sepharose column (GE Healthcare) washed with three column volumes of 20 mM Tris–HCl pH 8.0, 1 M ammonium sulfate. Bound N-acetylneuraminate lyase was eluted using a decreasing gradient of ammonium sulfate concentration to 20 mM Tris–HCl pH 8.0. Size-exclusion chromatography was employed as the final purification step using a HiLoad 16/60 Superdex 200 column (GE Healthcare) with 20 mM Tris–HCl pH 8.0 buffer. All purification steps were carried out at 277 K. N-Acetylneuraminate lyase was concentrated using a 30 kDa molecular weight cutoff centricon (Millipore).

To determine the protein concentration after each purification step, the Bradford assay (Bradford, 1976 ▶) was used. A lactate dehydrogenase coupled assay was implemented to measure the catalytic activity of N-acetylneuraminate lyase for the reverse reaction, as described previously (Devenish & Gerrard, 2009 ▶).

2.2. Sequence analysis of bacterial N-acetylneuraminate lyase  

A multiple protein sequence alignment was performed between N-acetylneuraminate lyase from S. aureus subspecies aureus MW2 (NP_645109) and four additional bacterial species [Aggregatibacter aphrophilus NJ8700 (YP_003007048), Clostridium botulinum D strain 1873 (ZP_04863430), Gemella hamemolysans M341 (ZP_08259625), Mannheimia haemoylytica serotype A2 strain Bovine (ZP_05988340.1)] as well as dihydrodipicolinate synthase from S. aureus subspecies aureus 71193 (YP_006195476) and E. coli strain K12 (NP_416973) (extracted from http://www.ncbi.nlm.hih.gov, protein accession numbers in parentheses). This alignment was performed using the program MUSCLE (Edgar, 2004 ▶), with manual editing.

2.3. Crystallization of N-acetylneuraminate lyase  

Crystallization studies were initially conducted using a 10 mg ml−1 preparation of methicillin-resistant S. aureus N-acetylneuraminate lyase in 20 mM Tris–HCl pH 8.0. Initial protein crystallization trials were performed at the CSIRO node of the Bio21 Collaborative Crystallization Centre (C3; http://www.csiro.au/c3/) using The PACT Suite and The JCSG+ Suite crystal screens (Newman et al., 2005 ▶, 2008 ▶) at 281 and 293 K as previously described (Burgess, Dobson, Dogovski et al., 2008 ▶; Dobson, Atkinson et al., 2008 ▶; Voss et al., 2009 ▶). Crystal screens were performed using the sitting-drop vapour-diffusion method with droplets consisting of 150 nl protein solution and 150 nl reservoir solution. Several of The JCSG+ Suite screen conditions produced crystals with a needle-like morphology at 293 K (Fig. 3 ▶).

Figure 3.

Figure 3

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Crystals of methicillin-resistant S. aureus N-acetylneuraminate lyase. The scale bar indicates 0.5 mm.

Optimization of the initial hits was carried out using the hanging-drop vapour-diffusion method. The following conditions were varied for optimization: 1.7–2.2 µl of reservoir solution [The JCSG+ Suite condition H7: 25%(w/v) PEG 3350, 200 mM ammonium sulfate, 100 mM bis-tris pH 5.5] and 2.0 µl or 2.5 µl of protein solution (10 mg ml−1, in 20 mM Tris–HCl pH 8.0) were equilibrated against 1 ml of reservoir solution in 24-well Linbro plates (Hampton Research) at 281 and 293 K. These conditions produced crystals in most wells.

2.4. Data collection and processing  

For X-ray data collection, methicillin-resistant S. aureus N-acetyl­neuraminate lyase crystals were briefly soaked in cryoprotectant solution containing reservoir solution made up to 20%(v/v) PEG 300 before being flash-cooled in liquid nitrogen. Intensity data were collected on the MX2 beamline at the Australian Synchrotron, Victoria, Australia (Fig. 4 ▶). Crystals that had been flash-cooled were mounted onto the beamline in a cold nitrogen stream at 110 K. The detector was positioned 169 mm from the crystal and data were collected in 0.5° steps for one 180° pass, with 90% attenuation and an exposure time of 2 s.

Figure 4.

Figure 4

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X-ray diffraction of methicillin-resistant S. aureus N-acetylneuraminate lyase.

Indexing and integration of the data were performed using the program iMOSFLM (Battye et al., 2011 ▶). The resulting intensity data were analysed using POINTLESS (Evans, 2006 ▶) from the CCP4 program suite (Winn et al., 2011 ▶) and XTRIAGE (Zwart et al., 2005 ▶). Scaling and data reduction were then performed using SCALA (Evans, 2006 ▶), also from CCP4. Molecular replacement was performed using the program Phaser (McCoy et al., 2007 ▶) as implemented in PHENIX (Adams et al., 2010 ▶). All relevant data-collection and processing parameters are given in Table 1 ▶. Images will be made available via the TARDIS server (Androulakis et al., 2008 ▶) when the structure is published.

Table 1. X-ray data-collection statistics for methicillin-resistant S. aureus N-acetylneur­aminate lyase.

Statistical values for the highest-resolution shells are given in parentheses. The Matthews coefficient and solvent content are based on eight monomers, with a molecular weight of 33 042.5 Da in the asymmetric unit.

Wavelength (Å) 0.95369
Number of images 360
Oscillations (°) 0.5
Space group P21
Unit-cell parameters (Å, °) a = 80.2, b = 108.6, c = 130.8 β = 89.9
Resolution (Å) 32.3–1.70 (1.79–1.70)
Observed reflections 823916 (91713)
Unique reflections 242175 (33054)
Completeness (%) 98.5 (92.4)
R merge † 0.101 (0.376)
R r.i.m. ठ0.118 (0.457)
R p.i.m. § 0.061 (0.254)
Mean I/σ(I) 8.0 (2.6)
Redundancy 3.4 (2.8)
Wilson B value (Å2) 15.5
Molecules per asymmetric unit 8
V M (Å3 Da−1) 2.16
Solvent content (%) 43
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†

R merge = Inline graphic Inline graphic.

‡

R r.i.m. = Inline graphic Inline graphic Inline graphic.

§

R p.i.m. = Inline graphic Inline graphic Inline graphic, where Ii (hkl) is the ith intensity measurement of reflection hkl, 〈I(hkl)〉 its average and N is the redundancy of a given reflection.

3. Results and discussion  

3.1. Expression and purification of N-acetylneuraminate lyase  

Purification of N-acetylneuraminate lyase was carried out using anion-exchange chromatography, hydrophobic interaction chromatography and size-exclusion chromatography, yielding approximately 32.8 mg of pure protein with a specific activity of 11 µmol min−1 mg−1 from 1.5 l of bacterial cell culture (Table 2 ▶). The purity of the sample was at least 95% as estimated by SDS–PAGE (Fig. 5 ▶).

Table 2. Purification of recombinant methicillin-resistant S. aureus N-acetylneuraminate lyase from a 1.5 l culture.

Purification step Total protein (mg) Specific activity (µmol min−1 mg−1) Total activity (µmol min−1) Yield (%)
Crude 408 6.5 2646 100
Anion exchange 177 8.2 1443 55
Hydrophobic interaction 34.4 19 669 25
Size exclusion 32.8 11 374 14
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Figure 5.

Figure 5

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Purification of recombinant methicillin-resistant S. aureus N-acetylneuraminate lyase. SDS–PAGE gel showing purification of methicillin-resistant S. aureus N-acetylneuraminate lyase. Lane 1, molecular-weight markers (in kDa); lane 2, crude cell lysate; lane 3, pooled fractions from the anion-exchange chromatography step; lane 4, pooled fractions from the hydrophobic interaction chromatography step; lane 5, post-size-exclusion chromatography.

3.2. Sequence analysis of bacterial N-acetylneuraminate lyase  

A multiple protein sequence alignment was carried out between N-acetylneuraminate lyase from S. aureus subspecies aureus MW2 and four additional bacterial species (A. aphrophilus NJ8700, C. botulinum D strain 1873, G. hamemolysans M341 and M. haemoylytica serotype A2 strain Bovine) as well as dihydro­dipicolinate synthase from S. aureus subspecies aureus 71193 and E. coli strain K12 (NP_416973). This was performed to investigate the homology of N-acetylneuraminate lyase in S. aureus subspecies aureus MW2 with N-acetylneuraminate lyase in representative Gram-positive and Gram-negative bacterial species. The sequence alignment (Fig. 6 ▶) illustrates conservation of the active-site Lys165 (amino-acid numbering corresponds to that of N-acetylneuraminate lyase from S. aureus subspecies aureus MW2). It is proposed that this active-site Lys165 binds the α-keto acid moiety of the substrate in both N-acetylneuraminate lyase and dihydrodipicolinate synthase throughout all species studied to date (Barbosa et al., 2000 ▶; Lawrence et al., 1997 ▶). Other active-site residues also associated with the binding of the α-keto acid moiety of putative substrates, including the GXXGE motif, Ser47 and Ser(Thr)48 (that form the respective second and third residues of the GXXGE motif), and Tyr137 were conserved within N-acetylneuraminate lyase and dihydrodipicolinate synthase between species (Dobson et al., 2004 ▶; Lawrence et al., 1997 ▶). In addition to the conservation of these integral active-site residues, preservation of a salt bridge, Glu50 to Lys256, linking the C-terminal three-helical cluster and the (β/α)8-barrel also seems to be highly conserved between species (Izard et al., 1994 ▶). Residue I210, which in dihydrodipicolinate synthases shows a conserved and strained non-planar peptide bond with the following serine residue (Dobson, Griffin et al., 2008 ▶), is also conserved, although the serine (S209 in E. coli dihydrodipicolinate synthase) is replaced by a glycine in N-acetylneuraminate lyase enzymes, suggesting that this strain may be released. Based on the sequence alignment, five core residues of the β-barrel (Ala11, Ile77, Tyr137, Lys165 and Gly189) are invariant across both the N-acetylneuraminate lyase and dihydrodipicolinate synthase enzymes within all species (Izard et al., 1994 ▶). This homology illustrates the importance of these residues and domains to the structure and function of the enzymes.

Figure 6.

Figure 6

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Sequence alignment of N-acetylneuraminate lyase from five bacterial species, as well as dihydrodipicolinate synthase from two bacterial species. Conserved residues are highlighted in pink. NAL denotes N-acetylneuraminate lyase and DHDPS denotes dihydrodipicolinate synthase.

3.3. Crystallization, data collection and processing of N-acetylneuraminate lyase  

As described in §2.3, several of The JCSG+ Suite screen conditions produced crystals with a needle-like morphology at 293 K (Fig. 3 ▶). In-house optimization of a successful condition (The JCSG+ Suite condition H7) selected from the high-throughput screening resulted in crystals that remained needle-like in morphology, although they were considerably larger in size. The best diffracting crystal was obtained from a 4.1 µl droplet consisting of 2.1 µl protein solution and 2.0 µl reservoir solution. An X-ray diffraction data set to a resolution of 1.70 Å was collected from this crystal. This resolution is comparable to that of the wild-type unliganded structure of N-acetyl­neuraminate lyase from H. influenzae (1.60 Å; PDB entry 1f6k; Barbosa et al., 2000 ▶) and the wild-type unliganded structure of N-acetylneuraminate lyase from E. coli (1.48 Å; PDB entry 3lbm; Chou et al., 2011 ▶).

Analysis of the intensity data using POINTLESS indicated that the crystals belong to the space group P21. XTRIAGE suggested that the data were twinned, with twin law Inline graphic. The l-statistic clearly suggests twinning with a value of 0.379 (untwinned = 0.5, perfect twin = 0.375). Initial phases were estimated by molecular replacement using the program Phaser. The monomer of H. influenzae N-acetyl­neuraminate lyase was used as the search model (PDB entry 1f6p; Barbosa et al., 2000 ▶) and the Matthews coefficient (Matthews, 1968 ▶) was calculated to be 2.16 Å3 Da−1 assuming eight monomers in the asymmetric unit, with a corresponding solvent content of 43%. The eight detected monomers appear to form two tetramers of similar monomer arrangement to that of other species of N-acetylneuraminate lyase. Molecular replacement with this search model using data scaled in P21 produced a final solution with a translation-function Z-score (TFZ) of 71.2. Refinement of the structure of methicillin-resistant S. aureus N-acetylneuraminate lyase is currently underway.

In summary, we present the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylneuraminate lyase from methicillin-resistant S. aureus subspecies aureus MRSA252. Understanding the structural biology of N-acetylneuraminate lyase in pathogenic species of bacteria, such as methicillin-resistant S. aureus, will provide us with the preliminary information necessary for future antimicrobial development.

Acknowledgments

We acknowledge the support and assistance of the friendly staff at the CSIRO-Collaborative Crystallization Centre at CSIRO Material Science and Engineering, Parkville, Melbourne and the MX beamline scientists at the Australian Synchrotron, Victoria, Australia. Parts of this research were undertaken at the MX2 beamline of the Australian Synchrotron, Victoria, Australia. Travel to the Australian Synchrotron was supported by the New Zealand Synchrotron Group. RCJD acknowledges the C. R. Roper Bequest for Fellowship support, the New Zealand Royal Society Marsden Fund for funding support, in part (contract UOC1013), and the US Army Research Laboratory and US Army Research Office under contract/grant No. W911NF-11-1-0481 for support, in part. HS acknowledges FY 2012 Researcher Exchange Program between the Japan Society for the Promotion of Science and the Royal Society of New Zealand for salary support. AOH acknowledges the United States National Science Foundation (NSF) award No. MCB-1120541. MDWG is the recipient of an Australian Research Council Post Doctoral Fellowship (project No. DP110103528). We especially thank Jackie Healy for her magnanimous technical support.

References

  1. Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221.
  2. Almagro-Moreno, S. & Boyd, E. F. (2009a). BMC Evol. Biol. 9, 118. [DOI] [PMC free article] [PubMed]
  3. Almagro-Moreno, S. & Boyd, E. F. (2009b). Infect. Immun. 77, 3807–3816. [DOI] [PMC free article] [PubMed]
  4. Androulakis, S. et al. (2008). Acta Cryst. D64, 810–814.
  5. Barbosa, J. A., Smith, B. J., DeGori, R., Ooi, H. C., Marcuccio, S. M., Campi, E. M., Jackson, W. R., Brossmer, R., Sommer, M. & Lawrence, M. C. (2000). J. Mol. Biol. 303, 405–421. [DOI] [PubMed]
  6. Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. W. (2011). Acta Cryst. D67, 271–281. [DOI] [PMC free article] [PubMed]
  7. Boyce, J., Cookson, B., Christiansen, K., Hori, S., Vuopiovarkila, J., Kocagoz, S., Oztop, A., Vandenbrouckegrauls, C., Harbarth, S. & Pittet, D. (2005). Lancet Infect. Dis. 5, 653–663. [DOI] [PMC free article] [PubMed]
  8. Bradford, M. M. (1976). Anal. Biochem. 72, 248–254. [DOI] [PubMed]
  9. Buchanan, C. L., Connaris, H., Danson, M. J., Reeve, C. D. & Hough, D. W. (1999). Biochem. J. 343, 563–570. [PMC free article] [PubMed]
  10. Burgess, B. R., Dobson, R. C. J., Bailey, M. F., Atkinson, S. C., Griffin, M. D. W., Jameson, G. B., Parker, M. W., Gerrard, J. A. & Perugini, M. A. (2008). J. Biol. Chem. 283, 27598–27603. [DOI] [PubMed]
  11. Burgess, B. R., Dobson, R. C. J., Dogovski, C., Jameson, G. B., Parker, M. W. & Perugini, M. A. (2008). Acta Cryst. F64, 659–661. [DOI] [PMC free article] [PubMed]
  12. Chambers, H. F. (1988). Clin. Microbiol. Rev. 1, 173–186. [DOI] [PMC free article] [PubMed]
  13. Chang, D.-E., Smalley, D. J., Tucker, D. L., Leatham, M. P., Norris, W. E., Stevenson, S. J., Anderson, A. B., Grissom, J. E., Laux, D. C., Cohen, P. S. & Conway, T. (2004). Proc. Natl Acad. Sci. USA, 101, 7427–7432. [DOI] [PMC free article] [PubMed]
  14. Chou, C.-Y., Ko, T.-P., Wu, K. J., Huang, K.-F., Lin, C.-H., Wong, C.-H. & Wang, A. H.-J. (2011). J. Biol. Chem. 286, 14057–14064. [DOI] [PMC free article] [PubMed]
  15. David, M. Z. & Daum, R. S. (2010). Clin. Microbiol. Rev. 23, 616–687. [DOI] [PMC free article] [PubMed]
  16. Devenish, S. R. A. & Gerrard, J. A. (2009). Biochem. Biophys. Res. Commun. 388, 107–111. [DOI] [PubMed]
  17. Dobson, R. C. J., Atkinson, S. C., Gorman, M. A., Newman, J. M., Parker, M. W. & Perugini, M. A. (2008). Acta Cryst. F64, 206–208. [DOI] [PMC free article] [PubMed]
  18. Dobson, R. C. J., Griffin, M. D., Devenish, S. R. A., Pearce, G. F., Huton, C. A., Gerrard, J. A., Jameson, G. B. & Perugini, M. A. (2008). Protein Sci. 17, 2080–2090. [DOI] [PMC free article] [PubMed]
  19. Dobson, R. C. J., Griffin, M. D. W., Jameson, G. B. & Gerrard, J. A. (2005). Acta Cryst. D61, 1116–1124. [DOI] [PubMed]
  20. Dobson, R. C., Valegård, K. & Gerrard, J. A. (2004). J. Mol. Biol. 338, 329–339. [DOI] [PubMed]
  21. Eaton, R. W. (1994). J. Bacteriol. 176, 7757–7762. [DOI] [PMC free article] [PubMed]
  22. Edgar, R. C. (2004). Nucleic Acids Res. 32, 1792–1797. [DOI] [PMC free article] [PubMed]
  23. Eriksen, N. H., Espersen, F., Rosdahl, V. T. & Jensen, K. (1995). Epidemiol. Infect. 115, 51–60. [DOI] [PMC free article] [PubMed]
  24. Evans, P. (2006). Acta Cryst. D62, 72–82. [DOI] [PubMed]
  25. Furuya, E. Y. & Lowy, F. D. (2006). Nature Rev. Microbiol. 4, 36–45. [DOI] [PubMed]
  26. Hare, R. (1982). Med. His. 26, 1–24. [DOI] [PMC free article] [PubMed]
  27. Hu, L., Umeda, A., Kondo, S. & Amako, K. (1995). J. Med. Microbiol. 42, 127–132. [DOI] [PubMed]
  28. Itzstein, M. von (2007). Nature Rev. Drug Discov. 6, 967–974. [DOI] [PubMed]
  29. Iwabuchi, T. & Harayama, S. (1998). J. Bacteriol. 180, 945–949. [DOI] [PMC free article] [PubMed]
  30. Izard, T., Lawrence, M. C., Malby, R. L., Lilley, G. G. & Colman, P. M. (1994). Structure, 2, 361–369. [DOI] [PubMed]
  31. Jeffcoat, R., Hassall, H. & Dagley, S. (1969a). Biochem. J. 115, 969–976. [DOI] [PMC free article] [PubMed]
  32. Jeffcoat, R., Hassall, H. & Dagley, S. (1969b). Biochem. J. 115, 977–983. [DOI] [PMC free article] [PubMed]
  33. Jeong, H. G., Oh, M. H., Kim, B. S., Lee, M. Y., Han, H. J. & Choi, S. H. (2009). Infect. Immun. 77, 3209–3217. [DOI] [PMC free article] [PubMed]
  34. Jevons, M. P. (1961). BMJ, 1, 124–125.
  35. Kefala, G., Evans, G. L., Griffin, M. D., Devenish, S. R., Pearce, F. G., Perugini, M. A., Gerrard, J. A., Weiss, M. S. & Dobson, R. C. (2008). Biochem. J. 411, 351–360. [DOI] [PubMed]
  36. Kluytmans, J., van Belkum, A. & Verbrugh, H. (1997). Clin. Microbiol. Rev. 10, 505–520. [DOI] [PMC free article] [PubMed]
  37. Lawrence, M. C., Barbosa, J. A., Smith, B. J., Hall, N. E., Pilling, P. A., Ooi, H. C. & Marcuccio, S. M. (1997). J. Mol. Biol. 266, 381–399. [DOI] [PubMed]
  38. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674. [DOI] [PMC free article] [PubMed]
  39. McDonald, M., Hurse, A. & Sim, K. N. (1981). Med. J. Aust. 2, 191–194. [DOI] [PubMed]
  40. Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497. [DOI] [PubMed]
  41. Mirwaldt, C., Korndörfer, I. & Huber, R. (1995). J. Mol. Biol. 246, 227–239. [DOI] [PubMed]
  42. Newman, J., Egan, D., Walter, T. S., Meged, R., Berry, I., Ben Jelloul, M., Sussman, J. L., Stuart, D. I. & Perrakis, A. (2005). Acta Cryst. D61, 1426–1431. [DOI] [PubMed]
  43. Newman, J., Pham, T. M. & Peat, T. S. (2008). Acta Cryst. F64, 991–996. [DOI] [PMC free article] [PubMed]
  44. Nouwen, J. L., Ott, A., Kluytmans-Vandenbergh, M. F., Boelens, H. A., Hofman, A., van Belkum, A. & Verbrugh, H. A. (2004). Clin. Infect. Dis. 39, 806–811. [DOI] [PubMed]
  45. Panlilio, A. L., Culver, D. H., Gaynes, R. P., Banerjee, S., Henderson, T. S., Tolson, J. S. & Martone, W. J. (1992). Infect. Control Hosp. Epidemiol. 13, 582–586. [DOI] [PubMed]
  46. Severi, E., Hood, D. W. & Thomas, G. H. (2007). Microbiology, 153, 2817–2822. [DOI] [PubMed]
  47. Speller, D. C., Johnson, A. P., James, D., Marples, R. R., Charlett, A. & George, R. C. (1997). Lancet, 350, 323–325. [DOI] [PubMed]
  48. Tuazon, C. U., Perez, A., Kishaba, T. & Sheagren, J. N. (1975). JAMA, 231, 1272. [PubMed]
  49. Tuazon, C. U. & Sheagren, J. N. (1974). J. Infect. Dis. 129, 725–727. [DOI] [PubMed]
  50. Vimr, E. R., Kalivoda, K. A., Deszo, E. L. & Steenbergen, S. M. (2004). Microbiol. Mol. Biol. Rev. 68, 132–153. [DOI] [PMC free article] [PubMed]
  51. Voss, J. E., Scally, S. W., Taylor, N. L., Dogovski, C., Alderton, M. R., Hutton, C. A., Gerrard, J. A., Parker, M. W., Dobson, R. C. J. & Perugini, M. A. (2009). Acta Cryst. F65, 188–191. [DOI] [PMC free article] [PubMed]
  52. Weinke, T., Schiller, R., Fehrenbach, F. J. & Pohle, H. D. (1992). Eur. J. Clin. Microbiol. Infect. Dis. 11, 985–989. [DOI] [PubMed]
  53. Wertheim, H. F., Melles, D. C., Vos, M. C., van Leeuwen, W., van Belkum, A., Verbrugh, H. A. & Nouwen, J. L. (2005). Lancet Infect. Dis. 5, 751–762. [DOI] [PubMed]
  54. Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242.
  55. Yu, V. L., Goetz, A., Wagener, M., Smith, P. B., Rihs, J. D., Hanchett, J. & Zuravleff, J. J. (1986). N. Engl. J. Med. 315, 91–96. [DOI] [PubMed]
  56. Zwart, P. H., Grosse-Kunstleve, R. W. & Adams, P. D. (2005). CCP4 Newsl. Protein Crystallogr. 43, contribution 7.

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