Structures of 5-Methylthioribose Kinase Reveal Substrate Specificity and Unusual Mode of Nucleotide Binding

The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp250-Glu252). In addition, the glycine-rich loop of the protein, analogous to the “Gly triad” in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp233 of the catalytic HGD motif, a novel twin arginine motif (Arg340/Arg341), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism. The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp250-Glu252). In addition, the glycine-rich loop of the protein, analogous to the “Gly triad” in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp233 of the catalytic HGD motif, a novel twin arginine motif (Arg340/Arg341), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism. Methionine is indispensable for cellular survival and is in high demand in proliferating cells. This essential amino acid plays critical roles in many ubiquitous cellular functions including protein synthesis, biological methylation, polyamine biosynthesis, as well as in the biosynthesis of the plant hormone ethylene and in some bacteria, quorum sensing. The biosynthesis of methionine is energetically costly, and the need for sufficient methionine has driven the evolution of methionine salvage pathways (1Sufrin J.R. Meshnick S.R. Spiess A.J. Garofalo-Hannan J. Pan X.Q. Bacchi C.J. Antimicrob. Agents Chemother. 1995; 39: 2511-2515Crossref PubMed Scopus (57) Google Scholar). Although the pathway is ubiquitous in almost all organisms, some metabolic variations are found in the pathways between mammals, plants, microbes, and certain parasitic protozoa. A key metabolic difference between mammals and prokaryotic pathogens is the absolute requirement for mtnK (2Sekowska A. Denervaud V. Ashida H. Michoud K. Haas D. Yokota A. Danchin A. BMC Microbiol. 2004; 4: 9Crossref PubMed Scopus (124) Google Scholar), which encodes 5-methylthioribose (MTR) 3The abbreviations used are: MTR, 5-methylthioribose; MTA, 5′-methylthioadenosine; APH(3′)-IIIa, 3′,5″-aminogylcoside phosphotransferase type IIIa; Ho-MAD, holmium multi-wavelength anomalous dispersion; AMPPNP, adenosine 5′-(β,γ-imido)triphosphate; AMPPCP, β,γ-methyleneadenosine 5′-triphosphate; PKA, cAMP-dependent protein kinase A.3The abbreviations used are: MTR, 5-methylthioribose; MTA, 5′-methylthioadenosine; APH(3′)-IIIa, 3′,5″-aminogylcoside phosphotransferase type IIIa; Ho-MAD, holmium multi-wavelength anomalous dispersion; AMPPNP, adenosine 5′-(β,γ-imido)triphosphate; AMPPCP, β,γ-methyleneadenosine 5′-triphosphate; PKA, cAMP-dependent protein kinase A. kinase (EC 2.7.1.100) (3Ferro A.J. Barrett A. Shapiro S.K. J. Biol. Chem. 1978; 253: 6021-6025Abstract Full Text PDF PubMed Google Scholar) for bacterial methionine salvage. MTR kinase is regulated by the environmental methionine level (4Tower P.A. Alexander D.B. Johnson L.L. Riscoe M.K. J. Gen. Microbiol. 1993; 139: 1027-1031Crossref PubMed Scopus (7) Google Scholar) and its expression enables organisms to grow on non-methionine sulfur sources such as MTR or 5′-methylthioadenosine (MTA) (5Riscoe M.K. Ferro A.J. Fitchen J.H. Antimicrob. Agents Chemother. 1988; 32: 1904-1906Crossref PubMed Scopus (29) Google Scholar, 6Sekowska A. Mulard L. Krogh S. Tse J.K. Danchin A. BMC Microbiol. 2001; 1: 15Crossref PubMed Scopus (26) Google Scholar, 7Sauter M. Cornell K.A. Beszteri S. Rzewuski G. Plant Physiol. 2004; 136: 4061-4071Crossref PubMed Scopus (43) Google Scholar). MTA is a byproduct and inhibitor of polyamine synthesis (8Sekowska A. Danchin A. BMC Microbiol. 2002; 2: 8Crossref PubMed Scopus (78) Google Scholar) and hence is toxic to cells and must be rapidly degraded. In various microbes, plants, and certain protozoa, MTA is degraded by MTA nucleosidase into MTR and adenine. MTR kinase then catalyzes the phosphorylation of MTR to MTR 1-phosphate, which is subsequently converted to methionine via a series of intermediates (8Sekowska A. Danchin A. BMC Microbiol. 2002; 2: 8Crossref PubMed Scopus (78) Google Scholar, 9Murphy B.A. Grundy F.J. Henkin T.M. J. Bacteriol. 2002; 184: 2314-2318Crossref PubMed Scopus (42) Google Scholar). In mammalian cells, however, the degradation of MTA and its conversion to MTR 1-phosphate is achieved in a single step by MTA phosphorylase (10Pegg A.E. Williams-Ashman H.G. Biochem. J. 1969; 115: 241-247Crossref PubMed Scopus (141) Google Scholar). This metabolic difference in the way MTA is removed has been explored and analogs of MTR, synthesized as pro-drugs, have been shown to selectively kill MTR kinase-containing organisms with little effect on mammalian cells (5Riscoe M.K. Ferro A.J. Fitchen J.H. Antimicrob. Agents Chemother. 1988; 32: 1904-1906Crossref PubMed Scopus (29) Google Scholar, 11Gianotti A.J. Tower P.A. Sheley J.H. Conte P.A. Spiro C. Ferro A.J. Fitchen J.H. Riscoe M.K. J. Biol. Chem. 1990; 265: 831-837Abstract Full Text PDF PubMed Google Scholar). The absence of a mammalian homolog makes MTR kinase a good target for the design of novel antibiotics. The modern rational approach to new drug discovery includes structure-based drug design, which requires a detailed understanding of the structure of the target enzyme and its catalytic mechanism and substrate specificity. Thus, three-dimensional structures of MTR kinase with and without substrates are indispensable for the development of specific antibiotics targeting this enzyme. To this end, we present the first structural analysis of MTR kinase in its apo form and in complex with ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR. Structure Determination of MTR Kinase Using Ho-MAD Phasing—Bacillus subtilis MTR kinase (gi:37999472) was expressed, purified, and crystallized as described previously (12Ku S.Y. Yip P. Cornell K.A. Riscoe M.K. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 116-119Crossref PubMed Scopus (11) Google Scholar). The crystals were derivatized by soaking the crystals for 3 days in 2 mm HoCl3, 25% (w/v) PEG2000MME, 25% (v/v) ethylene glycol, 0.3 m sodium acetate, and 0.1 m Tris-HCl, pH 7.8. The holmium derivative was flash frozen in a nitrogen stream at 110 K, and a MAD dataset was collected around the LIII edge of Ho to 2.5 Å resolution at Station X8C, National Synchrotron Light Source (NSLS), Brookhaven National Laboratory. The peak and inflection data were processed using HKL2000 (13Otwinowski Z. Minor W. Methods in Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar), and the remote data were reprocessed at home using d*TREK (14Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1410) Google Scholar). Two pairs of Ho atoms in the asymmetric unit were readily located using SnB (15Weeks C.M. Blessing R.H. Miller R. Mungee R. Potter S.A. Rappleye J. Smith G.D. Xu H. Furey W. Z. Kristallographie. 2002; 217: 686-693Google Scholar). The CNS suite of programs was used for heavy atom refinement, MAD phasing, and density modification (16Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. P. J. M. Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar). The data and are in MAD phasing, and in a new was using Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar, J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: PubMed Scopus Google Scholar), and of structure were between CNS (16Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. P. J. M. Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar) and in J. Biol. 1999; PubMed Scopus Google Scholar) with data A.T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; PubMed Google Scholar). the structure was density for was in the site in complex with Ho S.Y. Smith G.D. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar). The complex S.Y. Smith G.D. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar) was used as the for the of MTR kinase the kinase the complex the complex the complex and the substrate MTR complex Structure Determination of Kinase and of were as described previously (12Ku S.Y. Yip P. Cornell K.A. Riscoe M.K. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 116-119Crossref PubMed Scopus (11) Google Scholar). The and the complexes were by soaking of crystals in 25% (w/v) PEG2000MME, 0.3 m sodium acetate, mm magnesium acetate, and mm or The complex was by soaking the crystals in 25% (w/v) PEG2000MME, 0.3 m sodium acetate, mm magnesium acetate, mm AMPPCP, and 2 mm MTR for 2 The substrate MTR was synthesized by of MTA as described previously PubMed Scopus Google Scholar). by to the structure of In this mm and 2 mm were to the protein to the as described for (12Ku S.Y. Yip P. Cornell K.A. Riscoe M.K. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 116-119Crossref PubMed Scopus (11) Google Scholar). to of the by the ADP, was observed in of the of the complexes were in 25% (v/v) ethylene glycol, 25% (w/v) PEG2000MME, 0.3 m sodium for and flash frozen at 110 K. data were collected using and and processed using d*TREK (14Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1410) Google Scholar). The and was using the in A. Methods Enzymol. PubMed Scopus Google Scholar) with the structure of the complex as the of structure were then between Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; PubMed Scopus Google Scholar) and using P. K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: PubMed Scopus Google Scholar) a of Two and were and the was using in the suite Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; PubMed Scopus Google Scholar, Methods Enzymol. PubMed Scopus Google Scholar, Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar). The subtilis structural of and have of and of with the protein in density with in of the In density was observed in the the that the of have was A was in but in the for the to a density of in the difference A was in of this The has its atoms that it is not present at for the is by the that its was to its to of of the the of the we have the at and have its A a is in A the density for a is that observed in The complex has of refinement, and structure for the MTR kinase structures are in A of the and in the structures is in the were using W.L. The DeLano Scholar). and structure of subtilis MTR kinase has a bilobal of a lobe and a lobe by a Although of the is in the structures and not be the density the fold as the of a protein The of MTR kinase is of by and in a The C-lobe is with and with between and and a at the of the with and with the of and form a with the the of in a is with that of a protein Thus, MTR kinase is found in bacteria, plants, and organisms, the protein has the bilobal fold in eukaryotic protein kinases. The structure of subtilis MTR kinase has in the asymmetric unit by The interactions in the are are between and and and and interactions between and its and and its and between the of and the of of MTR kinase, of is L. 2002; PubMed Scopus Google Scholar). of the protein structure data K. Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar) that this is the found in of all kinases. The structure of subtilis MTR kinase is with the of the enzyme of in not and in of MTR A. Plant Physiol. PubMed Google Scholar, K.A. Tower P.A. Riscoe M.K. Biochem. J. PubMed Scopus Google Scholar), as well as with the observed for MTR kinase M. Cornell K.A. Beszteri S. Rzewuski G. Plant Physiol. 2004; 136: 4061-4071Crossref PubMed Scopus (43) Google Scholar). of MTR structural of MTR kinase to the using L. C. J. Biol. 1993; PubMed Scopus Google Scholar) or A. PubMed Scopus Google Scholar) structural to kinase D. P. C. Xu Z. Structure Full Text Full Text PDF PubMed Scopus Google Scholar) and phosphotransferase type G.D. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) of and that MTR kinase has of and Å with kinase and APH(3′)-IIIa, MTR kinase is to many eukaryotic protein including kinase G. J. J. 1995; PubMed Scopus Google Scholar), cAMP-dependent protein kinase J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; PubMed Google Scholar), and kinase J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the structural the found in the of MTR kinase, kinase, and readily the structural variations that for the of the substrates the to and of MTR kinase that are to all of the structure of kinase, kinase, and that a structures are MTR kinase via interactions between kinase via interactions between that are to of MTR kinase is as a in the structure via G.D. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) between and the between and The structure of kinase, kinase, and the catalytic of many protein kinase that to as in the absence of G. J. J. 1995; PubMed Scopus Google Scholar, H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the site is between the ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR MTR kinase structures and the in the substrate binding be readily The nucleotide in the between the of the MTR kinase, interactions with as well as with the The of the nucleotide the in a of and the of is the is by the of to the amino nitrogen of the and the the nitrogen of to the nitrogen A to the to the nitrogen of the and the to the of the of the nucleotide This is observed in all but the resolution In all nucleotide the atoms of the of the nucleotide interact via interactions with the nitrogen of the which in a with the of site of subtilis MTR A and the interactions between the and the protein the and complexes and the and complexes In the nucleotide and protein are as with and atoms and In atoms are in and are shown as and The in are the of with the in The in A is not observed in the complex the to in A of the in are in In the between MTR and the protein to in A of the in are in The in in does not have a in all structures its to is between the some Å are but are shown in that are of the are The of is in A of and of of the site between the of MTR kinase is by the of interactions between the ribosyl moiety of the nucleotide and In PKA, nucleotide binding lobe between in the C-lobe of and the nucleotide have been as as interactions a the and lobe closure S. J. Biol. 2004; PubMed Scopus Google Scholar). The in is which is located on the C-lobe and with ribosyl of J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; PubMed Google Scholar). No in MTR kinase plays A with structure is that the protein into a single or the of lobe or is to structures with that lobe is that that the of lobe closure observed between and the various complexes is not the of but to the of the protein, is as subtilis MTR kinase in the absence of in a (12Ku S.Y. Yip P. Cornell K.A. Riscoe M.K. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 116-119Crossref PubMed Scopus (11) Google Scholar) with in the the in structural as a of the we have the structure of MTR kinase with and K. A. and P. L. in This structure in and that the plant enzyme has the as the apo and of the bacterial enzyme. soaking not and of subtilis with in in the the soaking this suggests that MTR kinase is in the and that without the was for binding or and this was by have the crystals to the soaking The of the and by soaking complexes that lobe of of the not have the of the protein complex to its into the the site of MTR kinase as well as the and the site is readily in all structures to current structural data suggests that protein kinases, but with APH(3′)-IIIa, does not lobe upon and The motif or is found between and in the of MTR The of MTR kinase has a motif the is not in the loop but located on to The is analogous to the in G.D. 2002; PubMed Scopus (42) Google Scholar) and the “Gly triad” motif found in many eukaryotic protein such as W. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) In protein and APH(3′)-IIIa, the to and the or of the nucleotide. protein interactions between the nucleotide and the loop are interactions between the and the nucleotide are found in subtilis MTR between the of and the of the and between the nitrogen of and the of the The is observed in A of the and the between the and is and The of the density of this in of structures and in of the and complexes in to or No is observed between MTR kinase and the of the nucleotide. This of interactions between the ribosyl moiety of the nucleotide and the protein is unique to MTR kinase and appears to lobe The of is not because the site for the the substrate MTR for nucleotide binding. In the series of MTR kinase structures the is in some structures but not in the density of the is in A of all structures and not in of the or structures the of the nucleotide in are in A the for the protein are for the The in nucleotide which is with the that the is in in A. the is it appears to the nucleotide the Thus, the of the is to nucleotide and the The DXE and are for MTR kinase A.J. Methods Enzymol. PubMed Scopus Google Scholar). of structures that the interactions between the protein and the the are in complex with and The and complexes of the magnesium is in all and complexes and This site appears to be in the and In is by and of the of the of the of the In the and is by the of the of the of the and of AMPPCP, and a The magnesium found in complexes is by the and of and the of density be found in the or AMPPCP-MTR complexes for This is probably because or AMPPNP, lacks the in the that appears to be to this The to the DXE motif in which and in the This motif appears analogous to the motif found in many In this motif is in the nucleotide complex C. A. J. Biochem. PubMed Scopus Google Scholar) and is for J. 2001; PubMed Scopus Google Scholar, S. S. A. PubMed Scopus Google Scholar). The DXE motif is not found in protein or the kinases. have a DFG motif, which the of the J. Biol. PubMed Scopus Google Scholar). In and APH(3′)-IIIa, for the of the DFG motif in or in is in or The in in or in is not of the DFG motif J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar) The of the DFG motif in MTR kinase is in with its as metabolic which does not complex for of kinase and the of its site the structure of the complex M. A. J. Biol. PubMed Scopus Google Scholar), suggests that the DXE motif be kinase, MTR kinase, has a binding motif, a of the DXE motif in kinase has been to be in The of the in kinase is as it is in the The HGD and MTR substrate MTR in the between the of MTR kinase and is located the nucleotide In the of MTR between the and via with the form and hence the MTR is of the and of are found as by not In the the MTR the with the to the and of the the The is by a with and by interactions with Asp233 of the HGD motif and and of the twin arginine motif found in MTR the and of MTR form with Asp233 The HGD motif in MTR kinase is analogous to the motif, as of the eukaryotic protein and with the of the motif Asp233 in MTR the catalytic J. Biol. PubMed Scopus Google Scholar). The of this motif in MTR typically the of the DFG motif and is to substrate and in protein J. Biol. PubMed Scopus Google Scholar). This is not in MTR kinase, which lacks the DFG of the HGD motif in MTR kinase is or plays a in to be determined. The and MTR moiety of the MTR is by of the and and form a that to be in substrate specificity. are not all MTR kinase structure of a plant MTR kinase suggests that the a K. A. and P. L. in In the subtilis structure this to the of the moiety at the the is and is in A of the and AMPPCP-MTR This many MTR analogs with at the have been shown to be substrates of MTR kinase (3Ferro A.J. Barrett A. Shapiro S.K. J. Biol. Chem. 1978; 253: 6021-6025Abstract Full Text PDF PubMed Google Scholar, M.K. Ferro A.J. Fitchen J.H. Antimicrob. Agents Chemother. 1988; 32: 1904-1906Crossref PubMed Scopus (29) Google Scholar). Although the of the does not with are the to the for MTR observed for the enzyme in many of bacteria and M. Cornell K.A. Beszteri S. Rzewuski G. Plant Physiol. 2004; 136: 4061-4071Crossref PubMed Scopus (43) Google Scholar, K.A. Tower P.A. Riscoe M.K. Biochem. J. PubMed Scopus Google Scholar, A.J. Methods Enzymol. PubMed Scopus Google Scholar). Thus, the of appears to be to and via the W-loop, of MTR analogs to the for mechanism of in a kinase is as or dissociative K. J.H. A.J. S.R. P.A. Biol. 2001; PubMed Scopus Google Scholar). mechanism for MTR kinase a of the of MTR to the of a with between the and the and between the and the The dissociative mechanism is with first to form a which then its to the of MTR to form MTR of the enzyme and is to between the of the structures are of a dissociative mechanism. for this is as is or into MTR kinase, is observed in the that MTR kinase without MTR as a is observed for a the of MTR in the The density for is not as good as that observed for AMPPCP-MTR in complexes This is probably because of the resolution and of the in the the of the is at Å interactions in this site be The MTR site is of a that is of dissociative that the enzyme has a dissociative mechanism. the catalytic Asp233 appears to the MTR in the to the of The to the to a the is observed in plant K. A. and P. L. in has been observed and the binding or of the nucleotide. The current observed in the complexes does not the for in the MTR 1-phosphate, between the and the nucleotide. mechanism that have not been observed to many protein that lobe closure to a for the the of nucleotide and of MTR, are we the of to and in the absence of MTR, instead of MTR, the the mechanism. The structure suggests that the to the enzyme to that is observed in many Chem. PubMed Scopus Google Scholar). MTR to the the then be to the of MTR to form MTR its in MTR Asp233 the by the of The MTR 1-phosphate, then be by the The MTR binding site that is of and the binding of the MTR be by the twin arginine motif, and because the moiety of MTR 1-phosphate, the of MTR interactions with the for MTR kinase structures structural that be to design the of MTR kinase are as the structures to that on substrate binding. are this suggests that many protein kinase and that the design of kinase drug need to target the of MTR kinase is sufficient to a that be the substrate is by the enzyme or in the methionine by using protein kinase that target the site of a kinase fold H. M. Biochem. PubMed Scopus Google as structural and the unique of the and MTR binding it be to the of MTR kinase without specificity. the unique of binding for MTR kinase via its DXE motif of of MTR kinase to essential protein found in the the enzyme site that a be by MTR kinase are by be to be by G. D. Smith for D. and J. for the at The for and and for on MTR with

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