The Vitamin K-dependent Carboxylase Has Been Acquired by Leptospira Pathogens and Shows Altered Activity That Suggests a Role Other than Protein Carboxylation

Abstract

Leptospirosis is an emerging infectious disease whose pathology includes a hemorrhagic response, and sequencing of the Leptospira interrogans genome revealed an ortholog of the vitamin K-dependent (VKD) carboxylase as one of several hemostatic proteins present in the bacterium. Until now, the VKD carboxylase was known to be present only in the animal kingdom (i.e. metazoans that include mammals, fish, snails, and insects), and this restricted distribution and high sequence similarity between metazoan and Leptospira orthologs strongly suggests that Leptospira acquired the VKD carboxylase by horizontal gene transfer. In metazoans, the VKD carboxylase is bifunctional, acting as an epoxidase that oxygenates vitamin K to a strong base and a carboxylase that uses the base to carboxylate Glu residues in VKD proteins, rendering them active in hemostasis and other physiologies. In contrast, the Leptospira ortholog showed epoxidase but not detectable carboxylase activity and divergence in a region of identity in all known metazoan VKD carboxylases that is important to Glu interaction. Furthermore, although the mammalian carboxylase is regulated so that vitamin K epoxidation does not occur unless Glu substrate is present, the Leptospira VKD epoxidase showed unfettered epoxidation in the absence of Glu substrate. Finally, human VKD protein orthologs were not detected in the L. interrogans genome. The combined data, then, suggest that Leptospira exapted the metazoan VKD carboxylase for some use other than VKD protein carboxylation, such as using the strong vitamin K base to drive a new reaction or to promote oxidative damage or depleting vitamin K to indirectly inhibit host VKD protein carboxylation. Leptospirosis is an emerging infectious disease whose pathology includes a hemorrhagic response, and sequencing of the Leptospira interrogans genome revealed an ortholog of the vitamin K-dependent (VKD) carboxylase as one of several hemostatic proteins present in the bacterium. Until now, the VKD carboxylase was known to be present only in the animal kingdom (i.e. metazoans that include mammals, fish, snails, and insects), and this restricted distribution and high sequence similarity between metazoan and Leptospira orthologs strongly suggests that Leptospira acquired the VKD carboxylase by horizontal gene transfer. In metazoans, the VKD carboxylase is bifunctional, acting as an epoxidase that oxygenates vitamin K to a strong base and a carboxylase that uses the base to carboxylate Glu residues in VKD proteins, rendering them active in hemostasis and other physiologies. In contrast, the Leptospira ortholog showed epoxidase but not detectable carboxylase activity and divergence in a region of identity in all known metazoan VKD carboxylases that is important to Glu interaction. Furthermore, although the mammalian carboxylase is regulated so that vitamin K epoxidation does not occur unless Glu substrate is present, the Leptospira VKD epoxidase showed unfettered epoxidation in the absence of Glu substrate. Finally, human VKD protein orthologs were not detected in the L. interrogans genome. The combined data, then, suggest that Leptospira exapted the metazoan VKD carboxylase for some use other than VKD protein carboxylation, such as using the strong vitamin K base to drive a new reaction or to promote oxidative damage or depleting vitamin K to indirectly inhibit host VKD protein carboxylation. The vitamin K-dependent (VKD) 2The abbreviations used are: VKD, vitamin K-dependent; Gla, γ-carboxylated glutamic acid; ORF, open reading frame; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BES, N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid. carboxylase converts Glu residues to γ-carboxylated Glu (or Gla) residues in VKD proteins. In mammals this post-translational modification is required for VKD protein activities in a wide range of physiologies that includes hemostasis, calcium homeostasis, and growth control (1Berkner K.L. Runge K.W. J. Thromb. Haemost. 2004; 2: 2118-2132Crossref PubMed Scopus (145) Google Scholar). VKD protein carboxylation cannot occur in the absence of reduced vitamin K, as the carboxylase uses the energy of oxygenation of the reduced vitamin K to drive Glu carboxylation. Combined chemical modeling and biochemical studies indicate that the carboxylase deprotonates reduced vitamin K to generate a highly reactive vitamin K intermediate (K- in Fig. 1) with sufficient basicity (pKa ∼25) to deprotonate Glu (2Dowd P. Hershline R. Ham S.W. Naganathan S. Science. 1995; 269: 1684-1691Crossref PubMed Scopus (112) Google Scholar). This Glu carbanion is then carboxylated to Gla by the addition of CO2 while the vitamin K base is protonated to form the vitamin K epoxide product. The carboxylase, then, acts as both a vitamin K epoxidase and a Glu carboxylase and so is a bifunctional enzyme. The carboxylase is characterized by complex interactions with VKD proteins. All mammalian VKD proteins contain a high affinity carboxylase-binding site, usually a propeptide that is cleaved subsequent to carboxylation, that targets them to the carboxylase. Propeptide tethering results in processive carboxylation so that multiple Glu residues in the Gla domains of VKD proteins are all converted to Gla residues (3Stenina O. Pudota B.N. McNally B.A. Hommema E.L. Berkner K.L. Biochemistry. 2001; 40: 10301-10309Crossref PubMed Scopus (32) Google Scholar, 4Morris D.P. Stevens R.D. Wright D.J. Stafford D.W. J. Biol. Chem. 1995; 270: 30491-30498Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Full carboxylation transforms the Gla domain into a calcium binding module that promotes binding of the VKD proteins to cell surfaces that contain exposed anionic phospholipids or to hydroxyapatite. Carboxylation was first discovered in mammals; however, more recent studies show that carboxylase activity that depends upon vitamin K is also present in nonmammalian organisms, namely the marine snail Conus and Drosophila (5Bandyopadhyay P.K. Garrett J.E. Shetty R.P. Keate T. Walker C.S. Olivera B.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1264-1269Crossref PubMed Scopus (92) Google Scholar, 6Czerwiec E. Begley G.S. Bronstein M. Stenflo J. Taylor K. Furie B.C. Furie B. Eur. J. Biochem. 2002; 269: 6162-6172Crossref PubMed Scopus (45) Google Scholar, 7Li T. Yang C.T. Jin D. Stafford D.W. J. Biol. Chem. 2000; 275: 18291-18296Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 8Walker C.S. Shetty R.P. Clark K.A. Kazuko S.G. Letsou A. Olivera B.M. Bandyopadhyay P.K. J. Biol. Chem. 2001; 276: 7769-7774Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Conus venom contains short VKD peptides that paralyze prey by antagonizing neurotransmission (5Bandyopadhyay P.K. Garrett J.E. Shetty R.P. Keate T. Walker C.S. Olivera B.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1264-1269Crossref PubMed Scopus (92) Google Scholar, 6Czerwiec E. Begley G.S. Bronstein M. Stenflo J. Taylor K. Furie B.C. Furie B. Eur. J. Biochem. 2002; 269: 6162-6172Crossref PubMed Scopus (45) Google Scholar, 9Blandl T. Warder S.E. Prorok M. Castellino F.J. FEBS Lett. 2000; 470: 139-146Crossref PubMed Scopus (29) Google Scholar). The Conus VKD peptides and mammalian VKD proteins exhibit the same overall organization, i.e. they have a propeptide that is immediately upstream of the Gla domain. However, there is no obvious homology between Conus and mammals in either of these two domains. Nonetheless, interspecies cross-reactivity occurs: the Conus carboxylase can carboxylate a pentapeptide (FLEEL) derived from the mammalian Gla domain as well as a mammalian-derived propeptide-containing substrate (6Czerwiec E. Begley G.S. Bronstein M. Stenflo J. Taylor K. Furie B.C. Furie B. Eur. J. Biochem. 2002; 269: 6162-6172Crossref PubMed Scopus (45) Google Scholar). The Drosophila VKD carboxylase has also been shown to carboxylate FLEEL (7Li T. Yang C.T. Jin D. Stafford D.W. J. Biol. Chem. 2000; 275: 18291-18296Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Drosophila VKD proteins have not been identified, however, and so the functional consequences of VKD carboxylation in this organism are currently unknown. An important point regarding the Conus and Drosophila carboxylase orthologs is that in both cases they use vitamin K epoxidation to drive Glu carboxylation. Therefore, the fundamental role of vitamin K in these organisms is the same as that in mammals. Until now, the VKD carboxylase was known to be present only in multicellular organisms. Although vitamin K is found in some bacteria and plants, it is used there in respiration or photosynthesis, respectively, and VKD proteins are not present in these organisms. It was therefore a surprise when a VKD carboxylase ortholog was observed in the bacterial pathogen Leptospira interrogans (serovars Lai and Copenhageni) as a consequence of genome sequencing projects (10Nascimento A.L. Ko A.I. Martins E.A. Monteiro-Vitorello C.B. Ho P.L. Haake D.A. Verjovski-Almeida S. Hartskeerl R.A. Marques M.V. Oliveira M.C. Menck C.F. Leite L.C. Carrer H. Coutinho L.L. Degrave W.M. Dellagostin O.A. El-Dorry H. Ferro E.S. Ferro M.I. Furlan L.R. Gamberini M. Giglioti E.A. Goes-Neto A. Goldman G.H. Goldman M.H. Harakava R. Jeronimo S.M. Junqueira-de-Azevedo I.L. Kimura E.T. Kuramae E.E. Lemos E.G. Lemos M.V. Marino C.L. Nunes L.R. de Oliveira R.C. Pereira G.G. Reis M.S. Schriefer A. Siqueira W.J. Sommer P. Tsai S.M. Simpson A.J. Ferro J.A. J. 2004; PubMed Scopus Google Scholar, R. H. H. J. M. A. M.H. PubMed Scopus Google Scholar). Leptospira that include L. interrogans and L. a disease characterized by the of a wide of and by the for and in S. Leptospira and Scholar). The of are to the of a for the R. Haake D. B. R. M.H. J. The and Scholar). of is the hemorrhagic the role of the mammalian carboxylase in hemostasis, the of the VKD carboxylase ortholog some role in the of this therefore and the Leptospira ortholog as a first results showed that the Leptospira ortholog is an active that vitamin K but all other known VKD carboxylase orthologs it not show detectable carboxylation. The Leptospira was also from the mammalian ortholog in of epoxidation in the absence of Glu substrate. The of the VKD carboxylase in a pathogen and the of the acquired protein suggest that vitamin K epoxidation has been for a biochemical that a role in Leptospira and of the L. VKD VKD carboxylase was by the genome sequence of the region from the L. L. was used as to the in two a using and and two that used and either or The the and the the with base that and a The were into and The and from the were into The was with and and the was with the from and the and The contains the L. VKD carboxylase ortholog with by the was by the and were used to generate as K.L. McNally B.A. PubMed Scopus Google and were by a using or by activity of were then by the in Pudota B.N. K.W. Runge K.W. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). were with in the of by for all The were either or were first was to by and the was then with CHAPS, and was by the with for in one protein was than the Leptospira ortholog was not high and the more to be activity was in of CHAPS, vitamin K BES, and substrate or were with and then for as K.L. McNally B.A. PubMed Scopus Google Scholar). activity was using the same reaction that the was The epoxidase were by the addition of of were and by high as B.N. M. K.W. K.A. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google for the addition of a reaction for in vitamin K K epoxide was either by of the using the high or by the and that were then by were using a vitamin K epoxide the activity of the Leptospira the of protein was using a Leptospira ortholog were with a of human carboxylase of known as well as a control protein The was then in a using and substrate used as by using a Leptospira to the VKD from by using the human VKD carboxylase protein sequence as the the genome of the pathogen L. interrogans Lai R. H. H. J. M. A. M.H. PubMed Scopus Google a protein with a of and a of a by of from two Leptospira were the genome of L. interrogans was shown to a protein to the L. interrogans Lai for an (10Nascimento A.L. Ko A.I. Martins E.A. Monteiro-Vitorello C.B. Ho P.L. Haake D.A. Verjovski-Almeida S. Hartskeerl R.A. Marques M.V. Oliveira M.C. Menck C.F. Leite L.C. Carrer H. Coutinho L.L. Degrave W.M. Dellagostin O.A. El-Dorry H. Ferro E.S. Ferro M.I. Furlan L.R. Gamberini M. Giglioti E.A. Goes-Neto A. Goldman G.H. Goldman M.H. Harakava R. Jeronimo S.M. Junqueira-de-Azevedo I.L. Kimura E.T. Kuramae E.E. Lemos E.G. Lemos M.V. Marino C.L. Nunes L.R. de Oliveira R.C. Pereira G.G. Reis M.S. Schriefer A. Siqueira W.J. Sommer P. Tsai S.M. Simpson A.J. Ferro J.A. J. 2004; PubMed Scopus Google and a genome sequencing for the pathogen L. revealed the of a protein with sequence identity to the L. interrogans and using the L. interrogans Lai as the the protein base VKD carboxylases in a marine snail and in several mammals, with from to the sequence of the of the proteins In protein the similarity between the metazoan and Leptospira VKD as all Fig. (5Bandyopadhyay P.K. Garrett J.E. Shetty R.P. Keate T. Walker C.S. Olivera B.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1264-1269Crossref PubMed Scopus (92) Google Scholar, 6Czerwiec E. Begley G.S. Bronstein M. Stenflo J. Taylor K. Furie B.C. Furie B. Eur. J. Biochem. 2002; 269: 6162-6172Crossref PubMed Scopus (45) Google Scholar, 7Li T. Yang C.T. Jin D. Stafford D.W. J. Biol. Chem. 2000; 275: 18291-18296Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 8Walker C.S. Shetty R.P. Clark K.A. Kazuko S.G. Letsou A. Olivera B.M. Bandyopadhyay P.K. J. Biol. Chem. 2001; 276: 7769-7774Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, S.M. D. Stafford D.W. Science. PubMed Scopus Google Scholar). VKD for the carboxylase were not detected in the L. interrogans genome as no were found to the human VKD proteins protein protein protein Gla and The of the VKD carboxylase ortholog in Leptospira was VKD carboxylases were only known to be present in the animal kingdom (i.e. Fig. The with the L. interrogans as the also revealed VKD carboxylase orthologs in and in (i.e. from sequencing of bacterial from the K. A.L. D. J.A. D. S. K. O. J. J. R. H. Science. 2004; PubMed Scopus Google Scholar). This in bacteria is as of bacterial have been and VKD carboxylase orthologs are not detected in these In VKD carboxylase orthologs are not observed in plants, or bacteria the of a of from these This restricted distribution and the high of homology between these bacterial and metazoan orthologs strongly suggest horizontal of between M. with VKD carboxylase from a animal into the bacterial genome. This is by the of several other in the L. interrogans Lai and that are to mammalian i.e. and two domain and and and and and (10Nascimento A.L. Ko A.I. Martins E.A. Monteiro-Vitorello C.B. Ho P.L. Haake D.A. Verjovski-Almeida S. Hartskeerl R.A. Marques M.V. Oliveira M.C. Menck C.F. Leite L.C. Carrer H. Coutinho L.L. Degrave W.M. Dellagostin O.A. El-Dorry H. Ferro E.S. Ferro M.I. Furlan L.R. Gamberini M. Giglioti E.A. Goes-Neto A. Goldman G.H. Goldman M.H. Harakava R. Jeronimo S.M. Junqueira-de-Azevedo I.L. Kimura E.T. Kuramae E.E. Lemos E.G. Lemos M.V. Marino C.L. Nunes L.R. de Oliveira R.C. Pereira G.G. Reis M.S. Schriefer A. Siqueira W.J. Sommer P. Tsai S.M. Simpson A.J. Ferro J.A. J. 2004; PubMed Scopus Google Scholar, R. H. H. J. M. A. M.H. PubMed Scopus Google Scholar). The of these Leptospira proteins and to metazoan a to and Therefore, the L. VKD carboxylase ortholog was and as a first in the of these proteins. The Leptospira VKD a Leptospira VKD carboxylase ortholog was in these not carboxylase activity but can active when carboxylases from several and (6Czerwiec E. Begley G.S. Bronstein M. Stenflo J. Taylor K. Furie B.C. Furie B. Eur. J. Biochem. 2002; 269: 6162-6172Crossref PubMed Scopus (45) Google Scholar, 7Li T. Yang C.T. Jin D. Stafford D.W. J. Biol. Chem. 2000; 275: 18291-18296Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, D.A. A. C.T. Furie B. Furie B.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google are the Leptospira ortholog with a was and used to and the of this protein was by all of the Leptospira ortholog was in the is the same with mammalian and VKD carboxylases and is with the by the Leptospira gene Fig. The gene also a that not in in the Leptospira but be cell therefore for of human carboxylase with in a in the of the observed as The Leptospira however, was by that not occur and therefore not the of the Leptospira ortholog by The Leptospira all known VKD the epoxidation of vitamin K the energy required for the carboxylation and so epoxidation is a of carboxylation. therefore the Leptospira ortholog vitamin K to vitamin K The results showed that the Leptospira the human VKD carboxylase epoxidase activity that was was not observed in or in with an that activity was to the Leptospira or human The activity of the Leptospira epoxidase was with that of the human by epoxidase in with protein by a This showed that the Leptospira ortholog activity was but with that of the human Leptospira VKD epoxidase activity is to that of the human ortholog activity Leptospira in a new The human and Leptospira orthologs were both the with a showed not have upon showed that and human carboxylases have the same activity Pudota B.N. K.W. Runge K.W. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google and in the with an Leptospira ortholog showed that the not Leptospira activity not that was for Leptospira activity was epoxidation is by a base that for 1) and therefore is upon of the activities of the Leptospira and human orthologs several showed that the of activity to was for both not Leptospira K in carboxylase and the Leptospira ortholog were for carboxylase activity by into and epoxidase were in was using either or and human carboxylase was using mammalian-derived the of epoxidation observed was to that of carboxylation (i.e. an of for human carboxylase were with in the of However, the same was observed when the human carboxylase was in the absence of propeptide not is with results with carboxylase Furie B. C.T. Furie B.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and that the propeptide does not the of the two Leptospira VKD does not show detectable carboxylase activity activity activity Leptospira FLEEL Leptospira FLEEL FLEEL Leptospira in a new In to the human carboxylase, the Leptospira not exhibit detectable carboxylation of or FLEEL and from the observed with a control in have been detectable in the carboxylase Therefore, the of Leptospira epoxidase activity have a of the of the Leptospira ortholog was to that of the human carboxylase. The of detectable carboxylase activity therefore that the of Leptospira epoxidase activity is than carboxylase that the Leptospira ortholog is from the human carboxylase. The of detectable Leptospira carboxylase activity was to be of the VKD carboxylases show for of the binding in the and in substrate that contains a Glu is carboxylated Biochem. PubMed Scopus Google Scholar). more than one Glu are carboxylated more than with a but there is in substrate as peptides derived from VKD protein Gla domains are carboxylated with with that of peptides of with this all VKD carboxylases that have been and carboxylate the mammalian-derived FLEEL substrate with (6Czerwiec E. Begley G.S. Bronstein M. Stenflo J. Taylor K. Furie B.C. Furie B. Eur. J. Biochem. 2002; 269: 6162-6172Crossref PubMed Scopus (45) Google Scholar, 7Li T. Yang C.T. Jin D. Stafford D.W. J. Biol. Chem. 2000; 275: 18291-18296Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In a whose sequence K.A. Stenflo J. D.A. E. A. Begley G.S. Furie B.C. Furie B. Biochemistry. PubMed Scopus Google is from is carboxylated by both Conus and mammalian carboxylases with that are to that of the FLEEL substrate (6Czerwiec E. Begley G.S. Bronstein M. Stenflo J. Taylor K. Furie B.C. Furie B. Eur. J. Biochem. 2002; 269: 6162-6172Crossref PubMed Scopus (45) Google Scholar). for carboxylation by the human and Leptospira only the human showed detectable carboxylase activity The Leptospira then, to have or the to use epoxidase activity to carboxylation. The VKD for in the Leptospira VKD observed but not carboxylase, activity in the Leptospira ortholog the of this is regulated than the mammalian The epoxidase activity of VKD carboxylase has been shown to be regulated by VKD substrate such that epoxidation when Glu substrate is Furie B. C.T. Furie B.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This is not well the of an vitamin K base (K- in Fig. 1) that with other Glu is not for carboxylation. The of detectable carboxylase activity in the Leptospira ortholog the that epoxidation and carboxylation are not in this enzyme. therefore this by the of epoxidase activity in the or absence of VKD substrate. epoxidase activity was detectable when in the absence of Glu and the of Glu substrate epoxidase activity In contrast, the Leptospira showed epoxidase activity in the absence of substrate and was by the of Glu substrate. the Leptospira a from the human in that a high of epoxidation in the absence of VKD substrate. of the Leptospira VKD with VKD in a Glu sequence of the Leptospira VKD was with metazoan VKD carboxylases that have been shown either to exhibit carboxylase activity and or to contain VKD and carboxylase The metazoan VKD carboxylases all have a region of identity residues that has been by studies to be important to Glu binding Jin Stafford D.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the Leptospira VKD is in this divergence in residues and as important in the human VKD carboxylase. This of sequence identity and of Glu binding therefore carboxylase activity was not detected with the Leptospira carboxylases also have a of VKD substrate i.e. the site, but the Leptospira homology in this region is to this region is not well Leptospirosis results in characterized by and and recent genome sequencing of the pathogen L. interrogans R. H. H. J. M. A. M.H. PubMed Scopus Google revealed an ortholog of the VKD carboxylase, an observed only in multicellular organisms and known to be important to the Leptospira ortholog to have been acquired by horizontal and several of strongly suggest that the ortholog is and in Leptospira The ortholog is a that is is of an for proteins that includes one known to be in bacteria S. J. 1995; PubMed Google is observed in two other Leptospira and has an epoxidase activity to that of human VKD carboxylase the of functional In metazoans, the VKD carboxylase uses vitamin K epoxidation to carboxylate Glu residues in VKD proteins them The of the Leptospira however, is unknown. showed that Leptospira vitamin K epoxidation not in detectable Glu carboxylation and was i.e. in the absence of VKD substrate of the Leptospira with metazoan VKD carboxylases and divergence in a region of identity important for and a of the L. interrogans genome not the of VKD The combined then, suggest that the Leptospira VKD has been for some other role than VKD protein carboxylation. The of the VKD carboxylase by a pathogen the of this to Leptospira and the of the acquired suggest several is that the unfettered of the highly reactive intermediate (K- in Fig. 1) is to oxidative damage to the to for host the vitamin K base have been to drive a reaction that a to Leptospira is that the role of the Leptospira VKD epoxidase is in the of vitamin K. upon a of vitamin K, is from the and to all one VKD protein and therefore vitamin K and carboxylation for (1Berkner K.L. Runge K.W. J. Thromb. Haemost. 2004; 2: 2118-2132Crossref PubMed Scopus (145) Google Scholar). the of Leptospira in or present the host cell and therefore with host VKD protein by vitamin K to point with to the of the VKD ortholog is the has been for the same use in all VKD orthologs have also been observed in is a and in that are derived from an of organisms that in an K. A.L. D. J.A. D. S. K. O. J. J. R. H. Science. 2004; PubMed Scopus Google Scholar). 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Stafford D.W. 2004; PubMed Scopus Google and studies that this ortholog in with the Leptospira VKD as it does in mammals, be of The that the Leptospira ortholog has VKD epoxidase activity is of with to vitamin metazoan VKD carboxylase residues have been to there is no or homology to other proteins to to is the base that deprotonates reduced vitamin K to generate the highly reactive vitamin K intermediate recent studies indicate that the base is an than a as by a human carboxylase with all residues by is and the of the as well as the an Pudota B.N. K.W. Runge K.W. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). of the Leptospira with the metazoan VKD carboxylases only a of the of this is the of the vitamin of residues of carboxylase (i.e. all of the residues of in Fig. in a in both vitamin K and epoxidase to the that the vitamin K epoxidase domain the D.A. C.T. Furie B. Furie B.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). However, this region is not present in the Leptospira that not all of the epoxidase domain for the vitamin K epoxidase domain is the region the Fig. vitamin K is and therefore with of the to other with M.H. Stevens R.C. FEBS Lett. 2004; PubMed Scopus Google Scholar). are several of identity or similarity between the metazoan VKD carboxylases and the Leptospira and so the Leptospira be in to the epoxidase domain. the Leptospira be is in epoxidation and carboxylation are in metazoan VKD carboxylase showed a in epoxidation in the of VKD the Leptospira was a of the orthologs in the that the two in the metazoan VKD with