The Crystal Structure of the Escherichia coliMobA Protein Provides Insight into Molybdopterin Guanine Dinucleotide Biosynthesis

Abstract

The molybdenum cofactor (Moco) is found in a variety of enzymes present in all phyla and comprises a family of related molecules containing molybdopterin (MPT), a tricyclic pyranopterin with a cis-dithiolene group, as the invariant essential moiety. MPT biosynthesis involves a conserved pathway, but some organisms perform additional reactions that modify MPT. In eubacteria, the cofactor is often present in a dinucleotide form combining MPT and a purine or pyrimidine nucleotide via a pyrophosphate linkage. In Escherichia coli, the MobA protein links a guanosine 5′-phosphate to MPT forming molybdopterin guanine dinucleotide. This reaction requires GTP, MgCl2, and the MPT form of the cofactor and can efficiently reconstituteRhodobacter sphaeroides apo-DMSOR, an enzyme that requires molybdopterin guanine dinucleotide for activity. In this paper, we present the crystal structure of MobA, a protein containing 194 amino acids. The MobA monomer has an α/β architecture in which the N-terminal half of the molecule adopts a Rossman fold. The structure of MobA has striking similarity to Bacillus subtilis SpsA, a nucleotide-diphospho-sugar transferase involved in sporulation. The cocrystal structure of MobA and GTP reveals that the GTP-binding site is located in the N-terminal half of the molecule. Conserved residues located primarily in three signature sequence motifs form crucial interactions with the bound nucleotide. The binding site for MPT is located adjacent to the GTP-binding site in the C-terminal half of the molecule, which contains another set of conserved residues presumably involved in MPT binding. The molybdenum cofactor (Moco) is found in a variety of enzymes present in all phyla and comprises a family of related molecules containing molybdopterin (MPT), a tricyclic pyranopterin with a cis-dithiolene group, as the invariant essential moiety. MPT biosynthesis involves a conserved pathway, but some organisms perform additional reactions that modify MPT. In eubacteria, the cofactor is often present in a dinucleotide form combining MPT and a purine or pyrimidine nucleotide via a pyrophosphate linkage. In Escherichia coli, the MobA protein links a guanosine 5′-phosphate to MPT forming molybdopterin guanine dinucleotide. This reaction requires GTP, MgCl2, and the MPT form of the cofactor and can efficiently reconstituteRhodobacter sphaeroides apo-DMSOR, an enzyme that requires molybdopterin guanine dinucleotide for activity. In this paper, we present the crystal structure of MobA, a protein containing 194 amino acids. The MobA monomer has an α/β architecture in which the N-terminal half of the molecule adopts a Rossman fold. The structure of MobA has striking similarity to Bacillus subtilis SpsA, a nucleotide-diphospho-sugar transferase involved in sporulation. The cocrystal structure of MobA and GTP reveals that the GTP-binding site is located in the N-terminal half of the molecule. Conserved residues located primarily in three signature sequence motifs form crucial interactions with the bound nucleotide. The binding site for MPT is located adjacent to the GTP-binding site in the C-terminal half of the molecule, which contains another set of conserved residues presumably involved in MPT binding. molybdenum cofactor molybdopterin molybdopterin guanine dinucleotide dimethyl sulfoxide reductase The molybdenum cofactor (Moco)1 is an essential component of a diverse group of enzymes catalyzing important redox transformations in the global carbon, nitrogen, and sulfur cycles (1Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1470) Google Scholar, 2Kisker C. Schindelin H. Rees D.C. Annu. Rev. Biochem. 1997; 66: 233-267Crossref PubMed Scopus (439) Google Scholar). The Moco consists of a mononuclear molybdenum or tungsten coordinated by the dithiolene moiety of a family of tricyclic pyranopterin structures, the simplest of which is commonly referred to as molybdopterin (MPT) (3Rajagopalan K.V. Adv. Enzymol. 1991; 64: 215-290PubMed Google Scholar, 4Rajagopalan K.V. Johnson J.L. J. Biol. Chem. 1992; 267: 10199-10202Abstract Full Text PDF PubMed Google Scholar). In addition to MPT, various dinucleotide forms of the cofactor exist such as molybdopterin guanine dinucleotide (MGD) and molybdopterin cytosine dinucleotide (5Meyer O. Frunzke K. Tachil J. Volk M. Stiefel E.I. Molybdenum Enzymes, Cofactors and Chemistry. American Chemical Society, Washington, D. C.1993Google Scholar, 6Johnson J.L. Bastian N.R. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3190-3194Crossref PubMed Scopus (155) Google Scholar). The available crystal structures of enzymes containing Moco variants (7Chan M.K. Mukund S. Kletzin A. Adams M.W.W. Rees D.C. Science. 1995; 267: 1463-1469Crossref PubMed Scopus (530) Google Scholar, 8Romão M.J. Archer M. Moura I. LeGall J. Engh R. Schneider M. Hof P. Huber R. Science. 1995; 270: 1170-1176Crossref PubMed Scopus (457) Google Scholar, 9Schindelin H. Kisker C. Hilton J. Rajagopalan K.V. Rees D.C. Science. 1996; 272: 1615-1621Crossref PubMed Scopus (440) Google Scholar, 10Schneider F. Löwe J. Huber R. Schindelin H. Kisker C. Knäblein J. J. Mol. Biol. 1996; 263: 53-69Crossref PubMed Scopus (254) Google Scholar, 11Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 12Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (484) Google Scholar, 13McAlpine A.S. McEwan A.G. Shaw A.L. Bailey S. J. Biol. Inorg. Chem. 1997; 2: 690-701Crossref Scopus (152) Google Scholar, 14Czjzek M. Santos J.-P.D. Pommier J. Giordano G. Méjean V. Haser R. J. Mol. Biol. 1998; 284: 435-447Crossref PubMed Scopus (158) Google Scholar, 15Dias J.M. Than M.E. Humm A.E. Huber R. Bourenkov G.P. Bartunik H.D. Bursakov S. Calvete J. Caldeira J. Carneiro C. Moura J.J.G. Moura I. Romao M.J. Structure. 1999; 7: 65-79Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 16Dobbek H. Gremer L. Meyer O. Huber R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8884-8889Crossref PubMed Scopus (215) Google Scholar, 17Hu Y. Faham S. Roy R. Adams M.W.W. Rees D.C. J. Mol. Biol. 1999; 286: 899-914Crossref PubMed Scopus (117) Google Scholar) have revealed considerable structural diversity in the cofactor. The cofactor can exist in a mononucleotide as well as various dinucleotide forms, and either one or two copies of the different Moco variants ligate the metal. The dinucleotide forms of the cofactor appear to be present only in eubacteria. All known molybdoenzymes in Escherichia coli contain the bis-MGD form of the Moco that consists of two MGD molecules and a single molybdenum. Based on sequence similarities, all of these enzymes belong to the DMSOR family of Moco-containing enzymes (1Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1470) Google Scholar, 2Kisker C. Schindelin H. Rees D.C. Annu. Rev. Biochem. 1997; 66: 233-267Crossref PubMed Scopus (439) Google Scholar). The crystal structure ofRhodobacter sphaeroides DMSOR (9Schindelin H. Kisker C. Hilton J. Rajagopalan K.V. Rees D.C. Science. 1996; 272: 1615-1621Crossref PubMed Scopus (440) Google Scholar, 18Li H.-K. Temple C.A. Rajagopalan K.V. Schindelin H. J. Am. Chem. Soc. 2000; 32: 7673-7870Crossref Scopus (170) Google Scholar) revealed that the bis-MGD Moco is deeply buried in the enzyme. The molybdenum is ligated by the sulfurs of both dithiolene groups with an approximate 2-fold axis of symmetry passing through the metal. Only the molybdenum and some of its coordinating ligands are accessible from the solvent through an active site funnel. Genes involved in Moco biosynthesis have been identified in eubacteria, archaea, and eukarya. Besides the evolutionarily conserved common aspects of Moco biosynthesis (19Rajagopalan K.V. Neidhardt F.C. Escherichia coli and Salmonella typhimurium. American Society for Microbiology, Washington, D. C.1996: 674-679Google Scholar, 20Rajagopalan K.V. Biochem. Soc. Trans. 1997; 25: 757-761Crossref PubMed Scopus (64) Google Scholar), an additional step is present in eubacteria in which the MPT form of the cofactor is converted into a dinucleotide form (21Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar). In E. coli, the mobAB locus is responsible for the final step in Moco biosynthesis. E. coli MobA, also referred to as protein FA, consists of 194 amino acids and is responsible for the conversion of MPT and GTP to MGD (21Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar, 22Palmer T. Vasishta A. Whitty P.W. Boxer D.H. Eur. J. Biochem. 1994; 222: 687-692Crossref PubMed Scopus (48) Google Scholar). Proteins with homology to MobA are present in a variety of eubacteria (Fig. 1). It is currently unclear whether Moco-containing enzymes in all of these organisms utilize MGD or other dinculeotide forms of the cofactor. MobB is a GTP-binding protein, with weak intrinsic GTPase activity (23Eaves, D. J., Palmer, T., and Boxer, D. H. (1997)246, 690–697.Google Scholar). Reportedly, MobB is essential of but to the of active reductase T. C. Boxer D.H. Giordano G. Mol. 1996; PubMed Scopus Google of MobA from different conserved and residues in and in The signature are from a set of MobA to organisms to the MGD form of the This with the PubMed Scopus Google Scholar). structure as with J.M. Sci. 1994; PubMed Scopus Google Scholar) are The of cofactor into Moco-containing enzymes has been in some T. C. Boxer D.H. Giordano G. Mol. 1996; PubMed Scopus Google Scholar, Johnson J.L. Rajagopalan K.V. J.H. J. 1995; PubMed Google Scholar, A. Giordano G. F. J.H. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In all of the available crystal structures, the Moco is buried deeply the protein, that cofactor is to the final of protein In the of R. sphaeroides two forms of the have been H. M. T. J. 1994; PubMed Google Scholar). to be on its on and the other form is from active DMSOR these In the of molybdopterin cytosine from a variety of P. Meyer O. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar), and DMSOR from R. either or in the of MGD C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). some Moco-containing enzymes such as E. coli reductase a for Moco T. C. Boxer D.H. Giordano G. Mol. 1996; PubMed Scopus Google Scholar), such as DMSOR only MobA as single protein component cofactor and This is in the C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) on an in in which MobA only requires GTP, MgCl2, and the MPT form of the cofactor to efficiently active R. sphaeroides DMSOR from also that of MobA the presumably to binding of MGD to In to a of MGD biosynthesis and the of this cofactor into we have the crystal structure of E. with and bound The active site of MobA is located in a on the and the structure of MobA with GTP the MGD biosynthesis presumably in an on the of the enzyme that is well for binding MobA and as C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of MobA by of an enzyme a of a containing in The final of in The belong to the group with a and and contain one molecule in the The structure of MobA by single and a The by a crystal for in containing into a containing the and to and and the with an and and with the Enzymol. 1997; Scopus Google Scholar). The set to be of a but to only two one the and one a the and the set as the and three by A. 1990; Scopus Google Scholar). with E. G. Enzymol. 1997; PubMed Scopus Google Scholar) by solvent and to with D. 1996; PubMed Scopus Google Scholar). The in the is the is to a solvent of and this to the that be by solvent the to the to with A. R. Biol. 1999; PubMed Scopus Google Scholar) of the been set to the to of 194 residues in of the residues and the into the with the M. A. 1991; PubMed Scopus Google Scholar). The with G. A. E. D. 1997; PubMed Scopus Google Scholar), and molecules with All and the with the S. D. 1994; PubMed Scopus Google Scholar). to into MobA and a bound nucleotide be This to the that the of for with nucleotide binding. the of to the to this in a of GTP in the and of of and these from various and the with the Enzymol. 1997; Scopus Google Scholar). all of these and the as in the set for the The nucleotide in with GTP and and the is from a set from a crystal that in GTP and with and as for the The crystal structure of MobA by single with a crystal form group The structure has been to a of The of the is as by the the from and the of the of the residues found in the of the as in J.M. J. Google Scholar). and located in a are the only residues in of The final of the contains residues and a with an and and are and the N-terminal is presumably to and structure is the and is the of all the of the by its in to the in to is the of the structure by the of for is the of by the of the for of is the of for in a of of from groups of and are the and structure is as for of the from the of residues in the and of the as by in a is the and is the of all the of the by its in to the in to is the of the structure by the of for is the of by the of the for of is the of for and are the and structure is as for of the from the of residues in the and of the as by MobA has an α/β structure and is of a containing the of which is located the of the all are The is by three on either with the C-terminal located the N-terminal of the (Fig. and and the form the N-terminal half of MobA and have a in containing the Rossman but the structure of MobA is to the Rossman fold. additional is into the which with one in by the into the of the The MobA monomer has a with a the of the and the the of the The is and has a of It is and its N-terminal but is and in the the C-terminal the and has a of into the reveals a of the conserved and and are located in two of the signature sequence motifs is the of a which The of this the N-terminal of this to the adjacent in a from the of of the the of this is in an into the In all a to the of in the and that this has been The of be on the but the sulfur of is its is the of this of MobA from different organisms conserved (Fig. as a a to the found in a variety of the crystal structure of MobA, these residues form a that is and the and residues the conserved the N-terminal of residues in the the conserved and are located in this residues two conserved residues and located the C-terminal of a which into the C-terminal In the structure of MobA, all form located in the on the (Fig. This presumably the active site of The L. C. Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar) to the for structural of The with a of the nucleotide-diphospho-sugar transferase from Bacillus subtilis 1999; PubMed Scopus Google Scholar). two have and MobA has of is involved in the of the but its is The structural MobA and is of MobA can be with residues in with a of (Fig. has been with either or and the nucleotide the N-terminal of The binding site for the is located in the C-terminal half of the molecule and be by a molecule present in the The structural similarity and MobA is in this C-terminal different binding either a in or MPT in of containing the Rossman but these are related to MobA as by of the protein and H. and an a and form of the enzyme. The of the form is with a but the of an in the crystal and its structure the form by presumably to an In the the MobA protein is present as an (Fig. which has symmetry and is by symmetry in the The has of and contains a that its The of the is is the to a of is as the of the The on of the are by a The the are by residues is the to in and residues form a with the The of the is with as well as The is into the its monomer with other two the axis and two additional the 2-fold of of these only of the of the a of of the monomer is buried of the a be located in a one of the 2-fold symmetry axis (Fig. The identified as by an from the set a of to the of and the and solvent The revealed a with a of the the of the metal. The is coordinated with approximate by from monomer and two bound The are and The is a from the metal. The of is by the which contains the other the of is a on of the A. Biochem. PubMed Scopus Google Scholar) bound in the Temple and K. V. also on MobA but present in an with MobA, and the protein molybdenum and to the but is present as a in both the and for of the to into the MobA Only of the to the bound be in the The in well by the the addition of other or the from with GTP a and with either or Temple and Rajagopalan C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) have that either well in in The with in GTP and The cocrystal structure of MobA and has been to an of are the and the the two can be with a of The reveals that the nucleotide is bound in the (Fig. by the of the and the the is bound the and the moiety the of all three and the are to The purine is and located in sequence on one and in on the The amino group is to and (Fig. also forms a with its to of the In a buried molecule of the the on this of the The group the solvent and is involved in with The is located the of in to the of this which the the The groups in the form with from residues in sequence with the of and with the of The and is present in two and a with in only one of the and is the only forming interactions with the The is located the and and is coordinated by one from of these by of the conserved in sequence and by two The of the ligands is of an a site that is located the and the of The moiety is in an in which is to a the and The of the that GTP is bound with in the of MPT, and the of the groups of The cocrystal structure with bound GTP in the of a of in the moiety. is to that in the but in the other the is from the of the conserved residues and It that the residues in the to binding of the but as in the structure of the this is in all forms of of the reveals that the residues N-terminal and C-terminal to the from the bound This is another for the that in the of MPT, MobA is with GTP in a In E. coli, the of MobA Moco biosynthesis is to the MGD form of the cofactor by MPT and a reaction that to the of the and of GTP as In to the of this we have the crystal structure of MobA in the and of The binding site for GTP identified the N-terminal of in the cocrystal structure of MobA with The of the nucleotide binding site is the structural similarity of MobA and In the the nucleotide is bound in the as the GTP in MobA, has a different of the signature of MobA are located in the of the bound GTP, and residues in these crucial in nucleotide binding. of MobA GTP is by the amino group and the of the and the of and interactions binding of or pyrimidine and MPT. MGD is the form of the cofactor in E. coli, to be this and the structure a for nucleotide of a family of that and are This that some of these are involved in the of other dinucleotide forms of Moco such as molybdopterin cytosine dinucleotide has been in the C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), MobA is for the and of bis-MGD Moco in R. sphaeroides DMSOR in an in This only requires MobA, GTP, MgCl2, and the MPT form of the cofactor by of The step in the of MobA involves binding of and the MPT form of The crystal structure of MobA in with GTP has identified the GTP binding Based on the of GTP, we a for the which to the enzyme (Fig. to the bound additional is available in the active site of MobA, which presumably the MPT binding This site is located sequence which contains the conserved residues and the is by the conserved this is of a which This of the a that is located the of the active site the MPT binding the of MGD from the crystal structure of DMSOR H.-K. Temple C.A. Rajagopalan K.V. Schindelin H. J. Am. Chem. Soc. 2000; 32: 7673-7870Crossref Scopus (170) Google Scholar), we have MGD into the active site of The of MGD the and of the active site in The that the guanosine of MGD is in an as the guanosine in the cocrystal structure of MobA with GTP, for in the of the The pyrophosphate of MGD is in an with the is by as the the in both from the of The of the pyrophosphate in the group of into a as in one of the in the cocrystal structure of MobA with GTP and of MPT be by the of and which are both located in sequence The of the tricyclic MPT is as in Schindelin (9Schindelin H. Kisker C. Hilton J. Rajagopalan K.V. Rees D.C. Science. 1996; 272: 1615-1621Crossref PubMed Scopus (440) Google Scholar). the of the two to the binding of MPT. The is often by a which the in a as the the of MPT binding are the of residues in sequence and the conserved with its The of MobA presumably involves a by the of MPT on the of GTP with of the and of GTP as The residues in the only both GTP and MPT have been bound by The that the residues adjacent to the are from the bound nucleotide that structural in these which the to the and from the The conserved residues and adjacent to the with the the on the three from GTP and one from MPT. In to GTP the and we that the moiety of GTP adopts a in which the the and In the cocrystal structure with the and the in the moiety this the on the other be involved in the on the of the pyrophosphate group and be the crucial for the activity. be to the that is for GTP in the A. Rev. 1999; 32: PubMed Scopus Google Scholar), that in these GTP the and of this contain this and GTP only this has been into the active site by a protein K. L. A. F. A. Science. 1997; PubMed Scopus Google Scholar), the Temple and Rajagopalan C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) have that of MobA the of apo-DMSOR, that MobA is binding MGD with The crystal structure with this and the MGD binding site in the on the MobA the residues in the which form of sequence the MGD its pyrophosphate moiety as this the binding of MobA for MobA this of MGD into the of Moco-containing the other the binding be to for of MGD to the The cofactor in E. coli and R. sphaeroides DMSOR is present in the bis-MGD form in which the is ligated by the dithiolene groups of two MGD molecules as in all crystal structures of enzymes containing this of Moco J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (484) Google Scholar, 14Czjzek M. Santos J.-P.D. Pommier J. Giordano G. Méjean V. Haser R. J. Mol. Biol. 1998; 284: 435-447Crossref PubMed Scopus (158) Google Scholar, 15Dias J.M. Than M.E. Humm A.E. Huber R. Bourenkov G.P. Bartunik H.D. Bursakov S. Calvete J. Caldeira J. Carneiro C. Moura J.J.G. Moura I. Romao M.J. Structure. 1999; 7: 65-79Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 18Li H.-K. Temple C.A. Rajagopalan K.V. Schindelin H. J. Am. Chem. Soc. 2000; 32: 7673-7870Crossref Scopus (170) Google Scholar). is whether the MGD moiety by MobA contains the bound from the of the form of Moco as a or whether the is The is for the of MPT in to the structural of the (Fig. the dithiolene group is from the protein and is in with the protein, the of for the binding of MGD to the is present in the MGD by MobA, of the bis-MGD Moco the of molybdenum from the the other molybdenum is is as an additional step biosynthesis of It is also unclear whether MobA is involved in the of the bis-MGD cofactor and its to or whether the bis-MGD cofactor into the Based on the MobA crystal binding of bis-MGD the active site of the MobA monomer to be of The bis-MGD cofactor is to be in the active site of a MobA the MobA protein forms an in the crystal form this is of is the the of the MGD one of the be to to the of the bis-MGD form of the cofactor and its in the of the MobA The of the and the of the well for binding the bis-MGD cofactor. In the bis-MGD Moco to a MobA the a symmetry the protein and its The crystal structure of MobA with and GTP an of the of the step of Moco biosynthesis in E. coli, the of MGD from the MPT form of Moco and in MobA has been identified as the active site through of a cocrystal structure of the enzyme with located in three signature sequence motifs are crucial for binding of GTP in the N-terminal half of sequence the of MobA presumably forms an important with MPT, which is bound in the C-terminal half of the that the on the pyrophosphate group, and this a crucial in the of Schneider for and the of the and we for in the of