The Crystal Structure of 1-D-myo-Inosityl 2-Acetamido-2-deoxy-α-D-glucopyranoside Deacetylase (MshB) from Mycobacterium tuberculosis Reveals a Zinc Hydrolase with a Lactate Dehydrogenase Fold

Mycothiol (1-d-myo-inosityl 2-(N-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two β-sheets and two α-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD+ co-factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB. Mycothiol (1-d-myo-inosityl 2-(N-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two β-sheets and two α-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD+ co-factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB. It is estimated that there are currently 2.2 billion people infected with Mycobacterium tuberculosis (TB) 1The abbreviations used are: TBtuberculosisMSH1-d-myo-inosityl 2-(N-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranosideMcamycothiol S-conjugate amidaseInsinositol.1The abbreviations used are: TBtuberculosisMSH1-d-myo-inosityl 2-(N-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranosideMcamycothiol S-conjugate amidaseInsinositol. worldwide, leading to ∼2 million deaths annually (1Dye C. Scheele S. Dolin P. Pathania V. Raviglione M.C. JAMA (J. Am. Med. Assoc.). 1999; 282: 677-686Crossref PubMed Scopus (2701) Google Scholar, 2WHO Global Tuberculosis Control. WHO, Geneva, Switzerland1998: 1-3Google Scholar). To compound the urgency of this situation, 2% of TB clinical isolates display resistance to the common anti-TB medications, isoniazid and rifampicin (3Amaral L. Viveiros M. Kristiansen J.E. Trop. Med. Int. Health. 2001; 6: 1016-1022Crossref PubMed Scopus (68) Google Scholar). The latter of these two drugs was the one most recently introduced, in 1968. Clearly, the lack of new anti-TB drugs is a significant problem since the frequency of antibiotic-resistant TB is growing. To develop new anti-TB drugs, we have sought novel metabolic pathways or metabolic intermediates used by the bacteria. One such potential target is mycothiol (1-d-myo-inosityl 2-(N-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranoside, MSH or AcCys-GlcN-Ins) (Fig. 1), the reducing agent exclusively present in the order actinomycetes, to which TB belongs (4Newton G.L. Arnold K. Price M.S. Sherrill C. Delcardayre S.B. Aharonowitz Y. Cohen G. Davies J. Fahey R.C. Davis C. J. Bacteriol. 1996; 178: 1990-1995Crossref PubMed Google Scholar). This thiol is proposed to have a role similar to that of glutathione in controlling the levels of cellular reactive oxygen species, as an enzyme cofactor and as a potential reactant used for antibiotic removal from the bacteria (5Misset-Smits M. van Ophem P.W. Sakuda S. Duine J.A. FEBS Lett. 1997; 409: 221-222Crossref PubMed Scopus (55) Google Scholar, 6Norin A. Van Ophem P.W. Piersma S.R. Persson B. Duine J.A. Jornvall H. Eur. J. Biochem. 1997; 248: 282-289Crossref PubMed Scopus (47) Google Scholar, 7Newton G.L. Unson M.D. Anderberg S.J. Aguilera J.A. Oh N.N. delCardayre S.B. Av-Gay Y. Fahey R.C. Biochem. Biophys. Res. Commun. 1999; 255: 239-244Crossref PubMed Scopus (84) Google Scholar, 8Newton G.L. Av-Gay Y. Fahey R.C. Biochemistry. 2000; 39: 10739-10746Crossref PubMed Scopus (145) Google Scholar). Loss of mycothiol in mycobacteria is associated with slow growth and increased sensitivity to both reactive oxygen species and antibiotics (9Rawat M. Newton G.L. Ko M. Martinez G.J. Fahey R.C. Av-Gay Y. Antimicrob. Agents Chemother. 2002; 46: 3348-3355Crossref PubMed Scopus (163) Google Scholar). tuberculosis 1-d-myo-inosityl 2-(N-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranoside mycothiol S-conjugate amidase inositol. tuberculosis 1-d-myo-inosityl 2-(N-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranoside mycothiol S-conjugate amidase inositol. The biosynthetic pathway of mycothiol involves four steps: 1) production of 1-d-myo-inosityl-2-acetamido-2-deoxy-α-d-glucopyranose (GlcNAc-Ins) using the glycosyltransferase coded for by Rv0486 (MshA) (10Newton G.L. Koledin T. Gorovitz B. Rawat M. Fahey R.C. Av-Gay Y. J. Bacteriol. 2003; 185: 3476-3479Crossref PubMed Scopus (73) Google Scholar), 2) deacetylation of GlcNAc-Ins by MshB to produce 1-d-myo-inosityl 2-amino-2-deoxy-α-d-glucopyranoside (GlcN-Ins) (11Newton G.L. Av-Gay Y. Fahey R.C. J. Bacteriol. 2000; 182: 6958-6963Crossref PubMed Scopus (99) Google Scholar), 3) the addition of cysteine to the free amine of glucosamine in an ATP-dependent manner to produce Cys-GlcN-Ins, by MshC (Rv2130c) (12Sareen D. Steffek M. Newton G.L. Fahey R.C. Biochemistry. 2002; 41: 6885-6890Crossref PubMed Scopus (94) Google Scholar), and 4Newton G.L. Arnold K. Price M.S. Sherrill C. Delcardayre S.B. Aharonowitz Y. Cohen G. Davies J. Fahey R.C. Davis C. J. Bacteriol. 1996; 178: 1990-1995Crossref PubMed Google Scholar) acetylation of the amine of cysteine by acetyl-CoA (AcCys-GlcN-Ins) by MshD (Rv0819) (13Koledin T. Newton G.L. Fahey R.C. Arch. Microbiol. 2002; 178: 331-337Crossref PubMed Scopus (74) Google Scholar). We have solved the x-ray crystal structure of mycothiol deacetylase (MshB, TB gene Rv1170), an enzyme involved in the biosynthetic pathway of mycothiol. Recently, it was shown that Mycobacterial knockouts lacking MshB activity were more susceptible to reactive oxygen species but also more resistant to isoniazid (14Buchmeier N.A. Newton G.L. Koledin T. Fahey R.C. Mol. Microbiol. 2003; 47: 1723-1732Crossref PubMed Scopus (134) Google Scholar). The extensive sequence similarity of MshB to mycothiol S-conjugate amidase (Mca, TB gene Rv1082) has allowed us to build a faithful comparative molecular model of the latter. Mca may assist in the removal of antibiotics from the infectious bacteria by cleaving the S-conjugate formed by the reaction of MSH and an anti-biotic, thereby assisting in the export of the latter from the cell (8Newton G.L. Av-Gay Y. Fahey R.C. Biochemistry. 2000; 39: 10739-10746Crossref PubMed Scopus (145) Google Scholar, 15Newton G.L. Fahey R.C. Arch. Microbiol. 2002; 178: 388-394Crossref PubMed Scopus (154) Google Scholar). Protein Expression and Purification—The expression plasmid was used as described previously (11Newton G.L. Av-Gay Y. Fahey R.C. J. Bacteriol. 2000; 182: 6958-6963Crossref PubMed Scopus (99) Google Scholar). Protein was expressed overnight at room temperature by inducing with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside in Escherichia coli BL21(DE3) cells. Protein purification proceeded using the His6 tag present on the recombinant protein using a nickel-nitrilotriacetic acid affinity column and 150 mm imadazole elution buffer. Crystal Growth—Crystals of MshB were obtained by the vapor-diffusion method with a mother liquor consisting of 15% polyethylene glycol 4000, 50 mm Tris-HCl (pH = 8.0), 0.1 m Mg(NO3)2, 6% 1,6-hexanediol, and 10% ethylene glycol. A 1:2 ratio of protein solution (6 mg/ml) to mother liquor was mixed and left for vapor equilibration. Triclinic crystals formed after approximately 1 week at room temperature (Table I).Table ICrystallographic statistics for structure determinationCrystalNativeUNO3Space groupP1P1Cell dimensions a, b, c (Å)56.8, 74.0, 85.657.2, 73.8, 85.6 α, β, γ (Å)102.1, 108.2, 97.2101.6, 108.1, 97.4Wavelength (Å)1.00001.5418Resolution (Å)40—1.7 (1.79—1.70)aAll values in parentheses are for highest resolution shell40—2.5 (2.59—2.50)Completeness (%)96.3 (94.7)94.3 (91.5)Rsym(%)bRsym = ΣI — 〈I〉 /ΣI, where I is the observed intensity and 〈I〉 is the average intensity obtained from multiple observations of symmetry related reflections5.0 (29.6)7.6 (23.1)(I/σ(I))5.3 (2.2)9.0 (3.2)Redundancy3.9 (3.7)1.9 (1.8)Unique reflections134,27041,591Total reflections1,281,662470,788Figure of meritcTwo values are given, the first after substructure solution, the second after solvent flattening0.37—0.60 Number of protein atoms8364 Number of solvent atoms760Average B factors (protein/solvent) (Å2)22.2/31.5RcrystdRcryst = Σ∥Fo — Fc∥/Σ Fo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively0.194RfreeeRfree was calculate as for Rcryst with 5% of the data omitted from structural refinement0.228a All values in parentheses are for highest resolution shellb Rsym = ΣI — 〈I〉 /ΣI, where I is the observed intensity and 〈I〉 is the average intensity obtained from multiple observations of symmetry related reflectionsc Two values are given, the first after substructure solution, the second after solvent flatteningd Rcryst = Σ∥Fo — Fc∥/Σ Fo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectivelye Rfree was calculate as for Rcryst with 5% of the data omitted from structural refinement Open table in a new tab Data Collection and Heavy Atom Derivatives—A high resolution native data set was collected at beamline 8.3.1 at the Advanced Light Source in Berkeley, CA equipped with an ADSC Q210 detector. A heavy atom derivative was obtained by soaking crystals in 1 mm uranyl nitrate for 2 days. The derivative data set was collected on a Rigaku RU-H3R rotating anode generator equipped with a Rigaku R-AXIS IV++ image plate detector. The data were processed using MOSFLM (version 6.11) and scaled with SCALA (16Leslie, A. G. W. (1992) Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, No. 26, CCP4, York, UKGoogle Scholar, 17Evans P.R. Proceedings of the CCP4 Study Weekend on Data Collection and Processing. CCP4, York, UK1993: 114-122Google Scholar, 18Bailey S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar). Four heavy atom sites in the asymmetric unit were located using SOLVE via the single isomorphous replacement with anomalous scattering method (19Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1763-1775Crossref PubMed Scopus (78) Google Scholar). Solvent flattening and phase extension were done using RESOLVE (20Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1937-1940Crossref PubMed Scopus (281) Google Scholar). An initial model was built using aRP-wARP, which traced an initial 987 residues (21Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2559) Google Scholar). The structure was then subjected to iterative rounds of refinement with a maximum likelihood target using REFMAC (22Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13702) Google Scholar) and model fitting using XFIT (23McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2015) Google Scholar). Figures—All figures were produced using Pymol (24Delano W.L. The Pymol Molecular Graphics System. Delano Scientific, San Carlos, CA2002Google Scholar). Coordinates—The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 1Q74. Crystallography of MshB—The enzyme crystallized in the triclinic space group P1 with cell dimensions a = 56.8 Å, b = 74.0 Å, c = 85.6 Å, α = 102.1°, β = 108.2°, γ = 97.2°. There were four protein molecules in the unit cell (asymmetric unit), giving a solvent content of 50% (VM = 2.5 Å3/Da). The four molecules present were almost identical with a maximal pairwise Cα root mean square deviation of 0.30 Å over a minimum of 251 Cα atoms. All four molecules are missing two surface loops (minimally residues 100–103 and 164–167), which had untraceable electron density (see Fig. 2, a and b). Both of these regions are not located near the active site. Additionally, three of the four molecules are missing a surface loop minimally from residues 211–216. This loop is located proximal to the active site but is not expected to play a role in substrate binding or catalysis (see below). The one copy of MshB that contains this loop then represents the most complete model present for the protein. All four molecules are identical in the active site region and contain a catalytic zinc atom. Overall Structure of MshB—MshB consists of one large nine-stranded mixed β-sheet and one small three-stranded anti-parallel β-sheet (Fig. 2, a and b). The first five strands of the large β-sheet with the associated α-helices (α1 to α5) adopt a topology that closely resembles the Rossmann fold of lactate dehydrogenase (25White J.L. Hackert M.L. Buehner M. Adams M.J. Ford G.C. Lentz P.J. Smiley I.E. Steindel S.J. Rossmann M.G. J. Mol. Biol. 1976; 102: 759-779Crossref PubMed Scopus (174) Google Scholar). Several lines of evidence confirm that MshB is a zinc-binding protein (see below). Despite the diverse functions of lactate dehydrogenase (an oxidoreductase) and MshB (a zinc hydrolase), these enzymes adopt a similar fold. The three-dimensional structure of MshB does not have a related fold among the Zn2+-dependent metalloenzymes (see were by the but similar were L. C. J. Mol. Biol. PubMed Scopus Google The structure that there is a metal binding site of residues from the of 1 and the loop 1 to the and as as from the of In in the there are two water molecules that coordinate to the Zn2+ one of which is also to the carboxylate of a proposed catalytic (see Fig. of the substrate for MshB is 1-d-myo-inosityl a that is (see Fig. the active site and substrate binding of MshB is also and three carboxylate and three and one amide from the of four and and two residues and and molecules have similar of residues that form to the Biochem. PubMed Scopus Google Scholar). The of MshB to of a deacetylation reaction by MshB is similar to that by a cleaving a It was not completely that the of residues in the active site of MshB was similar to that of a The crystal of including and have been for a Data Bank accession and enzymes a metal by three protein two and a and a general base carboxylate that a water to the A identical of residues in the active site is in MshB deacetylase (Fig. The on the zinc are two and and an which from the in the The general base is a that is among Zn2+ S. We also two water molecules in the active both in the of the zinc and one near which is the nucleophilic water The other water is most displaced by an the water in the two water molecules to be and are not two with M. J. Mol. Biol. PubMed Scopus Google Scholar). The that both water molecules are present be by a of the bond in which both water molecules are to the expected of zinc J. Crystallogr. 1996; Google Scholar). was as the active site metal for three 1) metal data Newton and R. C. 2) a Zn2+ x-ray and 3) zinc a identical to that J. Mol. 1999; Google Scholar). The active site region of the enzyme is (Fig. A to this is which one of the active site (Fig. This is among related enzymes Rv1082) and may be of in the binding of since of these enzymes have this in common with (see below). of the similar of catalytic residues in the active sites of MshB and the zinc it is that the deacetylase also has a catalytic similar to that of the The involves nucleophilic of the with general base assistance of the carboxylate group of the on the of the In the general base is a The also has the role of general acid in the to the of the group of the In proposed catalytic (Fig. of of the substrate to MshB that the oxygen of the acetyl group the second water on the Zn2+ This the first water in an position for general nucleophilic of the of the acetyl The general base is the carboxylate of Asp-15. The tetrahedral would then have a oxygen atom that is by the Zn2+ and by the of to the of the group (GlcN-Ins) would be via the general acid of the group of Asp-15. This reaction be common to other as In other there is sequence similarity among the MshB in the region of the metal binding and the catalytic is an enzyme that the deacetylation of in The enzymes from a of species have a of that the metal binding and as as the catalytic general base (11Newton G.L. Av-Gay Y. Fahey R.C. J. Bacteriol. 2000; 182: 6958-6963Crossref PubMed Scopus (99) Google Scholar, T. M.L. J. Biol. 2002; PubMed Scopus Google Scholar). it has not been to substrate or to MshB. The of the deacetylase does not the does it with high Newton and R. C. Molecular of MSH in the active site of MshB two potential of the GlcNAc-Ins of the was for us to be of there were potential in the of the two most of the as the the catalytic activity of MshB by (11Newton G.L. Av-Gay Y. Fahey R.C. J. Bacteriol. 2000; 182: 6958-6963Crossref PubMed Scopus (99) Google Scholar). Clearly, there be of the with MshB. MshB amidase activity mycothiol cleaving the bond the to GlcN-Ins. The related high activity with mycothiol and deacetylase activity with G. and R. C. these two enzymes have Mca a role in the detoxification of which form with mycothiol (11Newton G.L. Av-Gay Y. Fahey R.C. J. Bacteriol. 2000; 182: 6958-6963Crossref PubMed Scopus (99) Google Scholar), and there is to that antibiotics are by an pathway Mca (9Rawat M. Newton G.L. Ko M. Martinez G.J. Fahey R.C. Av-Gay Y. Antimicrob. Agents Chemother. 2002; 46: 3348-3355Crossref PubMed Scopus (163) Google Scholar). G. and R. C. Mca may also be an target for TB drugs, and three-dimensional structure is of Mca by Open MshB and Mca sequence identity and over the first on this sequence a model for the catalytic of Mca was (Fig. T. J. M.C. Res. 2003; PubMed Scopus Google Scholar). The model was built to where the sequence identity for a The model similarity the two with active site residues including the metal binding site and the catalytic Mca has activity substrate be The major in the active sites is the of in where was in MshB. This may be in in which the in MshB with on the and the in Mca may the to it may in a or the with the could be for by with the of would most residues that the of and in MshB since this is where most of the sequence MshB and Mca and it was not to model this region Recently, the structure of a similar protein from was determined as of a structural T. Y. H. K. M. H. S. M. S. Protein 2003; PubMed Google Scholar). MshB and identity or sequence is residues in The role of is and the active site of the enzyme does not contain a metal atom. The enzymes a similar fold and active site and it is that has a similar role as a zinc the substrate (Fig. The of reactive oxygen and reactive intermediates is to Mycothiol to play a major role in mycobacteria G.L. Unson M.D. Anderberg S.J. Aguilera J.A. Oh N.N. delCardayre S.B. Av-Gay Y. Fahey R.C. Biochem. Biophys. Res. Commun. 1999; 255: 239-244Crossref PubMed Scopus (84) Google Scholar, M. Newton G.L. Ko M. Martinez G.J. Fahey R.C. Av-Gay Y. Antimicrob. Agents Chemother. 2002; 46: 3348-3355Crossref PubMed Scopus (163) Google Scholar, N.A. Newton G.L. Koledin T. Fahey R.C. Mol. Microbiol. 2003; 47: 1723-1732Crossref PubMed Scopus (134) Google Scholar) and in R. Biochem. J. 2003; PubMed Scopus Google Scholar). It that drugs MshB be to MSH but has shown that in M. tuberculosis (14Buchmeier N.A. Newton G.L. Koledin T. Fahey R.C. Mol. Microbiol. 2003; 47: 1723-1732Crossref PubMed Scopus (134) Google Scholar) and in Mycobacterium (9Rawat M. Newton G.L. Ko M. Martinez G.J. Fahey R.C. Av-Gay Y. Antimicrob. Agents Chemother. 2002; 46: 3348-3355Crossref PubMed Scopus (163) Google Scholar), of the gene does not MSH amidase, has GlcNAc-Ins deacetylase activity to a of MSH production a could be that both deacetylase then MSH production be a that Mca activity to the of antibiotics M. tuberculosis. The novel fold that MshB by contains in the of such drugs, the similarity of the active sites to a from which be We and at Advanced Light Source and Ko for J. T. and M. G. are to the for in data at the

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