The Mycobacterium tuberculosis lysAgene encodes the enzyme meso-diaminopimelate decarboxylase (DAPDC), a pyridoxal-5′-phosphate (PLP)-dependent enzyme. The enzyme catalyzes the final step in the lysine biosynthetic pathway converting meso-diaminopimelic acid (DAP) tol-lysine. The lysA gene of M. tuberculosis H37Rv has been established as essential for bacterial survival in immunocompromised mice, demonstrating thatde novo biosynthesis of lysine is essential for in vivo viability. Drugs targeted against DAPDC could be efficient anti-tuberculosis drugs, and the three-dimensional structure of DAPDC from M. tuberculosis complexed with reaction product lysine and the ternary complex with PLP and lysine in the active site has been determined. The first structure of a DAPDC confirms its classification as a fold type III PLP-dependent enzyme. The structure shows a stable 2-fold dimer in head-to-tail arrangement of a triose-phosphate isomerase (TIM) barrel-like α/β domain and a C-terminal β sheet domain, similar to the ornithine decarboxylase (ODC) fold family. PLP is covalently bound via an internal aldimine, and residues from both domains and both subunits contribute to the binding pocket. Comparison of the structure with eukaryotic ODCs, in particular with a di-fluoromethyl ornithine (DMFO)-bound ODC fromTrypanosoma bruceii, indicates that corresponding DAP-analogues might be potential inhibitors for mycobacterial DAPDCs. The Mycobacterium tuberculosis lysAgene encodes the enzyme meso-diaminopimelate decarboxylase (DAPDC), a pyridoxal-5′-phosphate (PLP)-dependent enzyme. The enzyme catalyzes the final step in the lysine biosynthetic pathway converting meso-diaminopimelic acid (DAP) tol-lysine. The lysA gene of M. tuberculosis H37Rv has been established as essential for bacterial survival in immunocompromised mice, demonstrating thatde novo biosynthesis of lysine is essential for in vivo viability. Drugs targeted against DAPDC could be efficient anti-tuberculosis drugs, and the three-dimensional structure of DAPDC from M. tuberculosis complexed with reaction product lysine and the ternary complex with PLP and lysine in the active site has been determined. The first structure of a DAPDC confirms its classification as a fold type III PLP-dependent enzyme. The structure shows a stable 2-fold dimer in head-to-tail arrangement of a triose-phosphate isomerase (TIM) barrel-like α/β domain and a C-terminal β sheet domain, similar to the ornithine decarboxylase (ODC) fold family. PLP is covalently bound via an internal aldimine, and residues from both domains and both subunits contribute to the binding pocket. Comparison of the structure with eukaryotic ODCs, in particular with a di-fluoromethyl ornithine (DMFO)-bound ODC fromTrypanosoma bruceii, indicates that corresponding DAP-analogues might be potential inhibitors for mycobacterial DAPDCs. meso-diaminopimelic acid meso-diaminopimelic acid decarboxylase pyridoxal 5′-phosphate SCID, severe combined immunodeficient phosphate-buffered saline with Tween 80 colony-forming unit 4-morpholineethanesulfonic acid Advanced Proton Source root mean square deviation alanine racemase α-difluoromethylornithine The final step in the bacterial lysine biosynthetic pathway is carried-out bymeso-DAP1decarboxylase (DAPDC), encoded by the lysA gene. DAPDC is a vitamin B6-dependent enzyme that stereospecifically converts meso-DAP to l-lysine (Scheme FS1). Like most enzyme-catalyzed decarboxylation reactions, the conversion of DAP to lysine is not reversible. The enzyme is of interest because of its importance in bacterial growth and survival. Lysine is required in protein biosynthesis and is essential for bacterial viability and development. The lysine precursor DAP itself is used as a structural cross-linking component of the peptidoglycan layer of Gram-negative, Gram-positive (except Gram-positivecocci), and mycobacterial cell walls (1Cummins C.S. Harris H. J. Gen. Microbiol. 1996; 14: 583-600Crossref Scopus (117) Google Scholar). DAP cross-links provide stability to the cell wall and confer resistance to intracellular osmotic pressure (2Strominger J.L. Fed. Proc. 1962; 21: 134-143PubMed Google Scholar). DAP can be synthesized by one or more of the following three different pathways: (i) the succinylase pathway, identified in all Gram-negative and Gram-positive bacteria, as well as Myobacterium tuberculosis; (ii) the dehydrogenase pathway, utilized by Bacillus sphaericus, Corynebacterium glutamicum, and Brevibacterium species (3Misono H. Nagasaki S. Soda K. Agric. Biol. Chem. 1986; 50: 1455-1460Google Scholar); and (iii) the acetylase pathway, which is limited to certain Bacillusspecies (4Sundharadas G. Gilvarg C. J. Biol. Chem. 1967; 242: 3983-3984Abstract Full Text PDF PubMed Google Scholar). Higher plants also produce lysine using a succinylase pathway (5Chatterjee S.P. Singh B.K. Gilvarg C. Plant Mol. Biol. 1994; 26: 285-290Crossref PubMed Scopus (15) Google Scholar). The presence of multiple biosynthetic pathways, at least in some bacteria, is probably an indication of the importance of DAP and lysine to bacterial survival. As the substrate and the reaction are not found in mammals, inhibitors of the enzyme may ultimately become leads for therapeutic intervention in bacterial infections (6McCann P.P. Pegg A.E. Pharmacol. Ther. 1992; 54: 195-215Crossref PubMed Scopus (234) Google Scholar). In Escherichia coli, the lysA gene is transcriptionally controlled by the LysR regulator protein; in the presence of lysine, transcription of the lysA gene is repressed (7Stagier P. Brone F. Richard F. Richard C. Patte J.C. J. Bacteriol. 1983; 156: 1198-1203Crossref PubMed Google Scholar). In contrast, M. tuberculosis does not apparently have a comparable LysR regulator, based on the lack of homologous sequences in the M. tuberculosis genomic sequence (8Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6660) Google Scholar). In M. tuberculosis, C. glutamicum, and Brevibacterium lactofermentum, the lysA gene is not in an operon as the second gene in an open reading frame withargS (arginyl-tRNA synthetase) (9Andersen A.B. Hansen E.B. Gene. 1993; 124: 105-109Crossref PubMed Scopus (13) Google Scholar, 10Sharp P.M. Mitchell K.J. Mol. Microbiol. 1993; 8: 200Crossref PubMed Scopus (8) Google Scholar, 11Marcel T. Archer J.A. Mengin-Lecreulx D. Sinskey A.J. Mol. Microbiol. 1990; 4: 1819-1830Crossref PubMed Scopus (16) Google Scholar, 12Oguiza J.A. Malumbres M. Eriani G. Pisabarro A. Mateos L.M. Martin F. Martin J.F. J. Bacteriol. 1993; 175: 7356-7362Crossref PubMed Google Scholar). In C. glutamicum the lysA gene is constitutively expressed (11Marcel T. Archer J.A. Mengin-Lecreulx D. Sinskey A.J. Mol. Microbiol. 1990; 4: 1819-1830Crossref PubMed Scopus (16) Google Scholar), and in the related organism B. lactofermentum thelysA gene is only weakly suppressed by lysine (12Oguiza J.A. Malumbres M. Eriani G. Pisabarro A. Mateos L.M. Martin F. Martin J.F. J. Bacteriol. 1993; 175: 7356-7362Crossref PubMed Google Scholar). Based on the evolutionary relationship between these three species of bacteria, we (13Pavelka Jr., M.S. Jacobs Jr., W.R. J. Bacteriol. 1996; 178: 6496-6507Crossref PubMed Scopus (151) Google Scholar) proposed that the expression of the lysA gene ofM. tuberculosis is probably constitutive. We show in this study that the lysA gene is essential for M. tuberculosis survival in an immunodeficient SCID (severe combined immunodeficient) mouse model, and we have determined the crystal structure of DAPDC in complex with the coenzyme pyridoxal 5′-phosphate (PLP) and the decarboxylation product lysine as well as DAPDC complexed with only lysine (binary complex). DAPDC is structurally very similar to eukaryotic ornithine decarboxylases (ODCs) (14Jackson L.K. Brooks H.B. Osterman A.L. Goldsmith E.J. Phillips M.A. Biochemistry. 2000; 39: 11247-11257Crossref PubMed Scopus (79) Google Scholar, 15Kern A.D. Oliveira M.A. Coffino P. Hackert M.L. Structure Fold. Des. 1999; 7: 567-581Abstract Full Text Full Text PDF Scopus (148) Google Scholar, 16Almrud J.J. Oliveira M.A. Kern A.D. Grishin N.V. Phillips M.A. Hackert M.L. J. Mol. Biol. 2000; 295: 7-16Crossref PubMed Scopus (131) Google Scholar) and, with the exception of a rotation of the C-terminal domain, to Bacillus stearothermophilus alanine racemase (AR) (17Shaw J.P. Petsko G.A. Ringe D. Biochemistry. 1997; 36: 1329-1342Crossref PubMed Scopus (258) Google Scholar). Although both DAPDC and ODCs carry-out similar decarboxylation reactions involving pyridoxal-5′-phosphate (PLP) as a cofactor, DAPDC is the only known amino acid decarboxylase that stereospecifically acts on a substrate carbon atom in d-configuration (SchemeFS1). The lysA mutant of M. tuberculosis, mc23026, was previously constructed by allelic exchange and has a deletion within the coding region of thelysA gene with an inserted γδ resolvase binding site (18Pavelka Jr., M.S. Jacobs Jr., W.R. J. Bacteriol. 1999; 181: 4780-4789Crossref PubMed Google Scholar). The mutant requires exogenous lysine supplementation at 1 mg/ml and can be complemented to protrophy by a copy of the wild-typelysA gene carried on the integrating vector pYUB651. In this work, we performed reversion analysis and were unable to isolate revertants from over 1010M. tuberculosisΔlysA cells. This established that the DAPDC activity can not be suppressed by any extragenic mutation and that the viability of the M. tuberculosis cells is dependent on this activity. Female SCID mice were bred at the animal facility of the Albert Einstein College of Medicine. The animals were maintained under barrier conditions and fed sterilized commercial mouse chow and water ad libitum. The M. tuberculosisstrains mc23026 (ΔlysA5::res) and mc23026 bearing pYUB651 (expressing the wild-typelysA gene) (13Pavelka Jr., M.S. Jacobs Jr., W.R. J. Bacteriol. 1996; 178: 6496-6507Crossref PubMed Scopus (151) Google Scholar), were grown in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween 80, 0.2% glycerol, and 1× ADS (0.5% bovine serum albumin, fraction V (Roche), 0.2% dextrose, and 0.85% NaCl) or on Middlebrook 7H10 or 7H11 solid medium (Difco) supplemented with 0.2% glycerol and 10% OADC (oleic acid, albumin, dextrose, and catalase; BD Biosciences). Cultures of the lysine auxotroph were supplemented with 1 mg/ml l-lysine (for both liquid and solid media), and 0.05% Tween 80 was added to solid medium. Liquid cultures were grown in 490-cm2 roller bottles (Corning) at 4–6 rpm. Plates were incubated for 3–6 weeks. Titered frozen stocks of bacteria were thawed and diluted appropriately in phosphate buffered saline containing 0.05% Tween 80 (PBST). The bacterial suspensions were plated at the time of injection to confirm viable counts. Intravenous injections were given via the tail vein. At various time points and three mice were for and the and were and in using a 80 The were diluted in and plated to the of colony-forming that mice were at in to the bacterial colony-forming by the mice to the colony-forming in the suspensions at the time of the bacterial at time the viable bacteria in the mice at containing thelysA gene was by with M. tuberculosis H37Rv genomic as the using the following and The was with and and the corresponding in the vector with a C-terminal cells were with the The cells were grown to at in containing of the cells were grown in supplemented with all amino and in Scholar). of lysA was with 1 and cells were growth for 4–6 at The cells were and in and containing 1 and inhibitors The cell was at with and the cell was at for 1 The was an and with of containing The DAPDC was from the using and to by on an DAPDC was against to and in at and DAPDC were at by in was carried with DAPDC DAPDC incubated with DAP PLP at and DAPDC of DAPDC was only in the of DAPDC supplemented with at within in of containing of lysine combined with of well against of well containing and were for in containing PLP to of the and from a crystal were at three using an on at the Advanced Source of the in were in a in of of a of and from were on using the The were using 1997; PubMed Scopus Google Scholar), and were with 1997; PubMed Scopus Google Scholar). The were with of the and or its J. Mol. Biol. PubMed Scopus Google Scholar) the presence of a dimer or a in the for were by 1997; PubMed Scopus Google Scholar) in the unit with a dimer in the unit Chem. PubMed Scopus Google Scholar), and J. Biol. 1999; PubMed Scopus Google Scholar) was used to the and protein in an of of for the in the of with in P. Biol. 1996; PubMed Scopus Google Scholar) in of for of protein in the The was to T. J.A. J.C. Proc. Mol. Biol. 1999; Scholar) for The of the and of the with the exception of a of amino that were in the the well the of the the from the using analysis and with the was and using Mol. Biol. 1999; PubMed Scopus Google Scholar). from the all of the residues of DAPDC could be the and using J. Biol. 1999; PubMed Scopus Google Scholar). final of was of and with E.J. Biol. 1997; PubMed Scopus Google Scholar) against the at the of the PLP phosphate was in the water were added of and for the complexed lysine was in binding of the dimer was not in the and for in for the for by in for the for by in for the for by in for the for by in for the for by for in and for in is is of final in for the for by for in and for in is is of in a and for and ternary DAPDC in for the in for the in for the in for the in for the in for the from from between subunits based on and against and are in the in for the from R. R. A. Scopus Google based on and against E.J. Biol. 1997; PubMed Scopus Google Scholar). and are in the in a The structure of DAPDC complexed with PLP and lysine was by with Biol. 1999; PubMed Scopus Google Scholar) using the final of the complex as a were using the P. B. Biol. PubMed Scopus Google Scholar). for both PLP and for both were in the to any of and in a final of for the The lysine mc23026 or the complemented mutant were cells SCID mice by tail and of three mice were at 1 and At the of viable bacteria was determined in the and of the The lysine mutant was from or not in the of the SCID mice, the complemented In both the and the the of viable bacteria by three of in the of the of viable bacteria in the was only one of The mice given the complemented M. tuberculosis mutant within the mice the M. tuberculosis mutant not any and for the of the have that mice can an with the M. auxotroph with the as for the of the mutant in the and of the SCID mice not In we the of reversion of thelysA by the mutant in the presence of lysine to of and in The of cultures and over cells from both cultures viable that the lysA deletion mutant does not and be suppressed by an extragenic The of the in and in vivo that DAPDC activity is essential for the viability of M. tuberculosis and that M. tuberculosis lysine from a We that targeted against DAPDC could be anti-tuberculosis and the of the three-dimensional structure ofM. tuberculosis The crystal structure of M. tuberculosis DAPDC confirms its classification as a fold type III dependent enzyme N.V. Phillips M.A. Goldsmith E.J. 4: PubMed Scopus Google Scholar). DAPDC has a fold similar to eukaryotic ODCs (14Jackson L.K. Brooks H.B. Osterman A.L. Goldsmith E.J. Phillips M.A. Biochemistry. 2000; 39: 11247-11257Crossref PubMed Scopus (79) Google Scholar, 15Kern A.D. Oliveira M.A. Coffino P. Hackert M.L. Structure Fold. Des. 1999; 7: 567-581Abstract Full Text Full Text PDF Scopus (148) Google Scholar, 16Almrud J.J. Oliveira M.A. Kern A.D. Grishin N.V. Phillips M.A. Hackert M.L. J. Mol. Biol. 2000; 295: 7-16Crossref PubMed Scopus (131) Google Scholar), and DAPDC also a stable head-to-tail of subunits with a deviation comparable with the for the structure and of the DAPDC subunits by 2-fold of domains is of residues a α/β of and The first residues are in domain and and of the The C-terminal domain residues and and and a by The structural domains are by and of the the β and were in the binding are by residues of both of the The active site is at the between the α/β domain of one and the β sheet domain of both from the α/β are in binding residues from the β sheet domain contribute to substrate between the and ternary complex are The only between the DAPDC complex the substrate and binding for structural using L. C. Full Text PDF PubMed Scopus Google Scholar) with eukaryotic ODCs, found in the biosynthetic pathway the decarboxylation of ornithine to and a of structural with from B. stearothermophilus sequence of known mycobacterial DAPDC eukaryotic ODCs with known and B. are in and with structural in the of amino acid sequence between eukaryotic ODCs and M. tuberculosis DAPDC least of the indicates which only with with The deviation can be to a rotation of the to the well α/β also Grishin N.V. Osterman A.L. Brooks H.B. Phillips M.A. Goldsmith E.J. Biochemistry. 1999; PubMed Scopus Google and structure for fold type III PLP dependent for M. and to to M. dimer glutamicum J.J. Oliveira M.A. Kern A.D. Grishin N.V. Phillips M.A. Hackert M.L. J. Mol. Biol. 2000; 295: 7-16Crossref PubMed Scopus (131) Google A.D. Oliveira M.A. Coffino P. Hackert M.L. Structure Fold. Des. 1999; 7: 567-581Abstract Full Text Full Text PDF Scopus (148) Google N.V. Osterman A.L. Brooks H.B. Phillips M.A. Goldsmith E.J. Biochemistry. 1999; PubMed Scopus Google J.P. Petsko G.A. Ringe D. Biochemistry. 1997; 36: 1329-1342Crossref PubMed Scopus (258) Google Myobacterium Myobacterium Bacillus in a of known fold type III PLP-dependent enzyme M. tuberculosis mouse T. of M. tuberculosis DAPDC with B. The rotation of the to the is The were carried by the corresponding are in The was using N. 1997; PubMed Scopus Google Scholar) and The sequence also show a of PLP binding from the mycobacterial to the eukaryotic ODCs and The containing the lysine that covalently to PLP via is in and eukaryotic ODCs, as is the to with the phosphate of the to be in and the and which are of the substrate binding R. L. B. P. Pegg A.E. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). with the exception of not in the structurally related alanine with its very sequence to the DAPDCs. Comparison of the at the dimer indicates that DAPDC or the most stable dimer the fold type III of known The of and the of crystal between related subunits in the crystal is that DAPDC is an structural for the dimer as the unit from the of a between of one of the dimer and of the are very in in is found only in mycobacterial is in all bacterial DAPDCs. also a via its to the PLP of the and is in all bacterial as well as in type III B6-dependent provide tuberculosis DAPDC is a stable DAPDC with an with a dimer in and the the subunits was by DAPDC was a P. B. J. PubMed Scopus Google Scholar), analysis that the DAPDC enzyme was S. A. T. C. T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). M. tuberculosis the crystal structure the a or the of a The active site of M. tuberculosis DAPDC is in a between the dimer with the PLP binding the C-terminal of the β of the α/β similar to ODCs (14Jackson L.K. Brooks H.B. Osterman A.L. Goldsmith E.J. Phillips M.A. Biochemistry. 2000; 39: 11247-11257Crossref PubMed Scopus (79) Google Scholar, 15Kern A.D. Oliveira M.A. Coffino P. Hackert M.L. Structure Fold. Des. 1999; 7: 567-581Abstract Full Text Full Text PDF Scopus (148) Google Scholar, 16Almrud J.J. Oliveira M.A. Kern A.D. Grishin N.V. Phillips M.A. Hackert M.L. J. Mol. Biol. 2000; 295: 7-16Crossref PubMed Scopus (131) Google Scholar). for PLP was in the of the ternary complex and the presence of a between and of PLP and between PLP and DAPDC are in The of the PLP phosphate with the of in the and of and also a with the of In the a the as the phosphate of PLP in the ternary In to the to the of PLP is by a to the of which in an with and the residues and residues and and with against the of the and are the of the and both are within of the of The of in the binding the of the of and as well as the of and with to the of to the of the dimer between the of and the of the The of with the of the This is in the eukaryotic and are and are by and alanine or In B. the is via and and are by and The on the of the by these could a in the of the different reactions in these In the the for reaction product lysine could be in binding In binding site the is very and and of the lysine In site the lysine is to the first its the in the binding site is not as as for site B. are with the the of the PLP with decarboxylation on this of the of domain of the in lysine binding with the of ODC to DAPDC that has been in mutation A.L. Grishin N.V. Phillips M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), which show a in substrate binding in The of lysine is by which in PLP binding via as As in the the and of lysine are to the by the internal of the substrate DAP based on the bound lysine have its in a to with the internal from the of the as well as with and the limited of the of the of the of the decarboxylation in DAPDC The structural between the DAPDC binding site and that of eukaryotic ODCs a related The of the decarboxylation reaction by ODCs has been (14Jackson L.K. Brooks H.B. Osterman A.L. Goldsmith E.J. Phillips M.A. Biochemistry. 2000; 39: 11247-11257Crossref PubMed Scopus (79) Google Scholar, A.L. Grishin N.V. Phillips M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The is that in ODCs the amino acid substrate ornithine is in an DAPDC the in the of with to the as an as well as of the of the the domain residues are for decarboxylation of acid decarboxylation reactions of fold type III PLP-dependent on the of the by Kern A.D. Oliveira M.A. Coffino P. Hackert M.L. Structure Fold. Des. 1999; 7: 567-581Abstract Full Text Full Text PDF Scopus (148) Google and that the reaction may an of the of the substrate K. K. M. Soda K. J. PubMed Scopus (15) Google Scholar). The of DAPDC with the and ODC (14Jackson L.K. Brooks H.B. Osterman A.L. Goldsmith E.J. Phillips M.A. Biochemistry. 2000; 39: 11247-11257Crossref PubMed Scopus (79) Google Scholar, N.V. Phillips M.A. Goldsmith E.J. 4: PubMed Scopus Google Scholar) of the indicates that given that is essential for M. tuberculosis could be a potential Although are known that one of the most used used to is α-difluoromethylornithine a that T. ODC N.V. Phillips M.A. Goldsmith E.J. 4: PubMed Scopus Google Scholar). In the crystal the with PLP as in the structure (14Jackson L.K. Brooks H.B. Osterman A.L. Goldsmith E.J. Phillips M.A. Biochemistry. 2000; 39: 11247-11257Crossref PubMed Scopus (79) Google Scholar), in is covalently bound to the of the binding site combined with an rotation of the to DAPDC the as the T. ODC the of the in has been proposed that in ODCs decarboxylation via the internal PLP by of a might a the R. L. B. P. Pegg A.E. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). a of a DAP might be of the is a probably with a rotation of which has its to the that a reaction can the the product the arrangement found in model, from a DAP as the bound in the T. shows that the is for a with of the decarboxylation reaction the of the that a DAP be stereospecifically at of We for for and on the B. R. C. and the for of at We for of the and for
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