Structural Studies of Molybdopterin Synthase Provide Insights into Its Catalytic Mechanism

Molybdenum cofactor biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea, and eukaryotes, including humans. Genetic deficiencies of enzymes involved in cofactor biosynthesis in humans lead to a severe and usually fatal disease. The molybdenum cofactor contains a tricyclic pyranopterin, termed molybdopterin, that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of molybdopterin is generated by molybdopterin synthase, which consists of a large (MoaE) and small (MoaD) subunit. The crystal structure of molybdopterin synthase revealed a heterotetrameric enzyme in which the C terminus of each MoaD subunit is deeply inserted into a MoaE subunit to form the active site. In the activated form of the enzyme, the MoaD C terminus is present as a thiocarboxylate. The present study identified the position of the thiocarboxylate sulfur by exploiting the anomalous signal originating from the sulfur atom. The structure of molybdopterin synthase in a novel crystal form revealed a binding pocket for the terminal phosphate of molybdopterin, the product of the enzyme, and suggested a binding site for the pterin moiety present in precursor Z and molybdopterin. Finally, the crystal structure of the MoaE homodimer provides insights into the conformational changes accompanying binding of the MoaD subunit. Molybdenum cofactor biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea, and eukaryotes, including humans. Genetic deficiencies of enzymes involved in cofactor biosynthesis in humans lead to a severe and usually fatal disease. The molybdenum cofactor contains a tricyclic pyranopterin, termed molybdopterin, that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of molybdopterin is generated by molybdopterin synthase, which consists of a large (MoaE) and small (MoaD) subunit. The crystal structure of molybdopterin synthase revealed a heterotetrameric enzyme in which the C terminus of each MoaD subunit is deeply inserted into a MoaE subunit to form the active site. In the activated form of the enzyme, the MoaD C terminus is present as a thiocarboxylate. The present study identified the position of the thiocarboxylate sulfur by exploiting the anomalous signal originating from the sulfur atom. The structure of molybdopterin synthase in a novel crystal form revealed a binding pocket for the terminal phosphate of molybdopterin, the product of the enzyme, and suggested a binding site for the pterin moiety present in precursor Z and molybdopterin. Finally, the crystal structure of the MoaE homodimer provides insights into the conformational changes accompanying binding of the MoaD subunit. Molybdenum cofactor (Moco) 1The abbreviations used are: Mocomolybdenum cofactorMPTmolybdopterinr.m.s.root mean squareE1ubiquitin-activating enzyme1The abbreviations used are: Mocomolybdenum cofactorMPTmolybdopterinr.m.s.root mean squareE1ubiquitin-activating enzyme biosynthesis is a phylogenetically conserved pathway present in all three kingdoms (1Rajagopalan K.V. Adv. Enzymol. 1991; 64: 215-290PubMed Google Scholar, 2Rajagopalan K.V. Biochem. Soc. Trans. 1997; 25: 757-761Crossref PubMed Scopus (64) Google Scholar, 3Schindelin H. Kisker C. Rajagopalan K.V. Adv. Protein Chem. 2001; 58: 47-94Crossref PubMed Scopus (66) Google Scholar). This pathway has been studied most extensively inEscherichia coli, where five mo loci have been discovered encoding one or more enzymes involved in Moco biosynthesis (2Rajagopalan K.V. Biochem. Soc. Trans. 1997; 25: 757-761Crossref PubMed Scopus (64) Google Scholar). Studies using several mo-deficient mutants in E. coli have elucidated the major steps during Moco biosynthesis. Although not all of the details of this pathway have been established, it can be divided into three common steps, where the second step is accomplished by the enzyme molybdopterin (MPT) synthase (4Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar). In humans, genetic defects in Moco biosynthesis result in Moco deficiency, a rare but severe disease accompanied by serious neurological symptoms including attenuated growth of the brain, untreatable seizures, dislocated ocular lenses, and mental retardation. Moco deficiency usually causes death in early infancy (5Johnson J.L. Rajagopalan K.V. Wadman S.K. Adv. Exp. Med. Biol. 1993; 338: 373-378Crossref PubMed Scopus (25) Google Scholar, 6Reiss J. Hum. Genet. 2000; 106: 157-163Crossref PubMed Scopus (97) Google Scholar). molybdenum cofactor molybdopterin root mean square ubiquitin-activating enzyme molybdenum cofactor molybdopterin root mean square ubiquitin-activating enzyme MPT synthase catalyzes the incorporation of the dithiolene moiety into precursor Z (4Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar) to form MPT (see Fig. 1 for structures). E. coli MPT synthase is composed of two subunits encoded by themoaD and moaE genes. In its active form, MoaD contains a thiocarboxylate at its C-terminal glycine. MPT synthase uses the thiocarboxylated C terminus as the sulfur donor during the synthesis of the dithiolene group. The formation of carbon-sulfur bonds is an integral process in the generation of both Moco and thiamin (7Begley T.P. Xi J. Kinsland C. Taylor S. McLafferty F. Curr. Opin. Chem. Biol. 1999; 3: 623-629Crossref PubMed Scopus (85) Google Scholar,8Rizzi M. Schindelin H. Curr. Opin. Struct. Biol. 2002; 12: 709-720Crossref PubMed Scopus (49) Google Scholar). The lack of mechanistic information about sulfur transfer chemistry as well as the biological importance of sulfur put great emphasis on understanding the sulfur transfer chemistry in these systems. A common step during thiamin and molybdopterin biosynthesis is the ATP-dependent transfer of sulfur froml-cysteine to yield a C-terminal glycine thiocarboxylate on ThiS and MoaD, respectively (9Leimkuhler S. Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 34695-34701Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 10Leimkuhler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 22024-22031Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 11Xi J. Ge Y. Kinsland C. McLafferty F.W. Begley T.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8513-8518Crossref PubMed Scopus (102) Google Scholar). For Moco biosynthesis, MoeB, the MPT synthase sulfurylase, converts the C-terminal carboxylate of MoaD into the thiocarboxylate with the aid of an IscS-like protein (10Leimkuhler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 22024-22031Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The C-terminal thiocarboxylate of MPT synthase and ThiS are the sulfur donors during either Moco or thiamin biosynthesis. In order for MoaD to interact with both MoeB and MoaE, MoaD must reversibly bind to either protein (Fig. 1a). This complex interaction is vital for the action of MPT synthase, where two MoaD subunits transfer the required sulfur atoms in two subsequent steps (12Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The structural basis for the transfer of MoaD is important in clarifying the enzymatic function of MPT synthase and the sulfur transfer cycle between MPT synthase and MoeB. The high resolution crystal structure of MPT synthase (Fig.1b) revealed a heterotetrameric molecule in which the two MoaE subunits dimerize and the MoaD subunits are located on opposite ends of the molecule with their C termini deeply inserted into each MoaE subunit (13Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar). MoaD adopts the same fold as ubiquitin despite the absence of detectable sequence homology, demonstrating a divergent evolutionary relationship between the MoaD-MoeB system and ubiquitin as well as the related protein modifiers SUMO and NEDD8 (Rub) and their activating (E1) enzymes. The MoaE subunit is characterized by an α/β hammerhead fold with an additional subdomain containing a four-stranded anti-parallel β-sheet. The active site of the enzyme is located in each MoaE subunit in close proximity to the MoaD C terminus (Fig. 1b), which is lined by conserved Lys, Arg, and His residues. While the majority of these residues originate from the proximal MoaE subunit, two residues (His-103′ and Arg-104′) are located in the distal MoaE subunit. Based on their phylogenetic conservation and their location in close proximity to the active site, these residues have been postulated to be important for the function of MPT synthase (13Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar). We present here the 1.9-Å resolution crystal structure of MPT synthase in a novel orthorhombic space group. This structure revealed a the MoaD C terminus that the binding site for the terminal phosphate group of the product of the In the structure of MoaE in the absence of MoaD at resolution from the crystal structure of a MoaE in which the residues have been the MPT synthase structure and the MoaD-MoeB three each of MoaE and MoaD are and these have been to the conformational changes in MoaD and the MoaE the site Finally, the position of the thiocarboxylate sulfur of MoaD has been identified anomalous In an accompanying (12Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (93) Google has been to study the of a of conserved MoaE residues in the MPT synthase with these the here the of MPT synthase and the of residues in its of activated MPT synthase as (13Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar). activated MoaD, MoaE, and the of MoaE as in the accompanying (12Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). of activated MPT synthase accomplished by of activated MoaD and The synthase a and the (13Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar). A crystal form of MPT synthase using activated MPT synthase with and as at a protein of The and their of The to the orthorhombic space group with of a and one in the to in that to this a structure by and this structure revealed that the MoaE C terminus of a of MoaE The of MoaE from a containing and at a protein of The to their of in and to the orthorhombic space group with of a and MoaE are present in the for the orthorhombic form of MPT synthase from at at the at and at at the for MoaE at a of of both in containing to A of for activated MPT synthase at a of on a with and an and using Enzymol. 1997; 276: PubMed Scopus Google Scholar). The of the orthorhombic crystal has an and of and respectively and in the The an of and in the resolution of The of the MoaE contains from a of in the resolution from to The and are and respectively and in the and the an of in the resolution (see Although the MoaE to the resolution to with where is and is the mean of all of the of the divided by its where is and is the mean of all of the of the divided by its in a The orthorhombic MPT synthase structure by using MPT synthase in the space group (13Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar) as the and the J. A. 157-163Crossref Scopus Google Scholar). The and using in the resolution from to and the space group identified during the The MoaE structure by using a MoaE homodimer as the The and with in the resolution from to with the Biol. 2001; PubMed Scopus Google Scholar, A. A. Biol. 2000; PubMed Scopus Google Scholar). of the three MoaE be located by but Y. Biol. 2001; PubMed Scopus Google and Biol. 2001; PubMed Scopus Google to the the information from the two used to the of the homodimer with A. Nat. Struct. Biol. 1999; PubMed Scopus Google Scholar) using structure to to containing residues of residues with five of these to the five of a of residues. of these with an MoaE the be located in the by and the used to and all of The orthorhombic form of MPT synthase at 1.9-Å the activated form of MPT synthase at and MoaE at all with the A. E. Biol. 1997; PubMed Scopus Google Scholar). for the orthorhombic form and MoaE with the and additional of the and of all additional residues accomplished with M. A. 1991; PubMed Scopus Google Scholar). For subsequent the S. Biol. PubMed Scopus Google Scholar). The anomalous of activated MPT synthase at The active site of MPT synthase with Biol. PubMed Scopus Google Scholar) using a of the sulfur in the MoaD activated MPT synthase in the crystal form, and to a resolution of with a of to the anomalous signal originating from sulfur The anomalous the position of the thiocarboxylate sulfur at the C terminus of The to the of the and is the and the sulfur of the (13Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google residues in the MoaD and MoaE subunits be identified with a and the This result the incorporation of the sulfur to be as by into the required for the resolution of is an for the formation of a complex for in the MoaD C which result in the anomalous The with a and a in the (Fig. at a the position of the is additional of the of the sulfur atom. The of the thiocarboxylate a with the group of the of the sulfur a with the of The interaction is responsible for the formation of the complex between the two subunits in which the of is to the MoaD C terminus an (13Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar). The formation of this complex is an of MPT synthase that the enzyme and on a of at this the position of the thiocarboxylate it is not that the MoaD C terminus conformational changes binding to yield the The structure of MPT synthase in the orthorhombic crystal form at 1.9-Å resolution to an of The residues of the MoaD subunit and residues and of the MoaE subunit, and of MoaE are in the structure of MPT synthase (13Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar). The of the as by the and In the has well with of all and residues in the most of the J. 1993; Google one in and two residues and in of these residues are located in in the of in to of where and are the and structure same as of the from in to the resolution from the of residues in the most and of the as by the of atoms as in where and are the and structure same as of the from in to the resolution the of residues in the most and of the as by the J. 1993; Google Biol. 1999; PubMed Scopus Google Scholar) as in in a The structure of the orthorhombic form of MPT synthase with the structure of MPT of the an of of conformational changes are present for residues and This structural to be by crystal between the orthorhombic and crystal this structural the The in both is in both with a in the orthorhombic crystal and are bonds in each of the in a of demonstrating that MoaE in both by a of is between the two crystal the in MoaE the of the MoaE is for the in the orthorhombic crystal form for the in the crystal A between the two MPT synthase crystal is the of a molecule (Fig. in the orthorhombic crystal form that a (Fig. in the crystal form from high of are located in the same pocket located in close proximity to the MoaD C The is by and and by three of the distal MoaE subunit are the of these bonds the of and of (Fig. and the is by the of with the binding pocket in the crystal form, and conformational changes in their In the orthorhombic is from the and the the to more for the and to a of this with its atom. A of three for the orthorhombic crystal form of MPT synthase including an at to or into these with of and of the not binding of either to the in all three a the of in a pocket in close proximity to the (Fig. this not be with of the it most of Z MPT in a their pterin be inserted deeply into the binding pocket present the MoaD C terminus The of this is a to the pterin moiety of either precursor Z or the of the conserved of the residues this pterin binding site are atoms originating from three of the and the MoaD C are the most for with the atoms of the The structure of the MoaE homodimer by in with with The in the are into three The in the by are to the that the atoms in this have and their to the is of the MoaE C termini at residues but one to the present in the In to both of MPT synthase, the residues in the from are in the In the all residues in this be and these residues form a structure that the active site. In two of the additional residues with the two MPT synthase are but a The structure of MoaE to of of including all to resolution The of the is as by the and In the has of and in the most in the and in the in and in in of the J. 1993; Google Scholar). The MoaE structure conformational changes with the MoaE subunits in the and orthorhombic MPT synthase The in is of residues of the MoaE with the residues of MoaE in MPT synthase The with the is the by and where as high as for are residues are from the the to the the majority of the structural changes are to the that the with MoaD in the MPT synthase (Fig. In this conformational changes of the of several residues and involved in with MoaD are The absence of MoaD this structural between the two MoaE and the of the atoms as by their (Fig. The most in are for residues which not major conformational but of the MoaD C in in the MoaE structure the of with residues at the terminus of this The conformational changes accompanying MoaD binding the of the two residues at and to as by the of their to interact with the MoaD C A in the position of for most of this in between the two residues. The of the MoaE in the absence of and in the of in the crystal form of MPT the pocket is not but contains a in five of the MoaE In of the the is in an where it with the of and to the molecule in the orthorhombic crystal form, these are by the position where the MoaD C terminus be located in MPT with the two MPT synthase the of and in MoaE are to the adopts the same as in the MPT In the MoaE subunit, the is present at the same position but not interact with either The binding pocket of the subunit is with the structure of the MoaD-MoeB complex Wuebbens M.M. Rajagopalan K.V. Schindelin H. 2001; PubMed Scopus Google are three MoaD crystal of these can be with of the atoms from MoaD in the two MPT synthase to the MoaD-MoeB complex and the orthorhombic MPT synthase the are between MoaD in the complex with MoeB and MoaD in the two MPT synthase these are small the MoaD C-terminal residues are of these residues large in the MoaD C termini to in the of the which the of the MoaD C termini in both The C terminus of MoaD in the absence of a protein is to be as has been for the C termini of ubiquitin S. J. Biol. PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google SUMO A. S. F. J. J. Biol. PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. S. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and ThiS C. Xi J. Begley T.P. Nat. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). conformational changes in MoaD are located in of the where between the two MPT MoaD three MoaD are at their with either MoaE or MoeB. of MoaD residues is by both MoaE and MoeB (Fig. and a of bonds in the two MPT synthase and in the MoaD-MoeB is present in the two a is in the MoaD-MoeB complex with the MPT synthase MPT synthase catalyzes the of precursor Z into MPT sulfur transfer from the MoaD thiocarboxylate. The of MPT synthase on the between the active form of MoaD the thiocarboxylate and the MoaE In both have to two of and to transfer the two sulfur atoms required for the of precursor Z to MPT The and of the MoaD with either MoaE or MoeB are for the in which precursor Z is to and the structural on these that at the of the MoaD is of in both protein as the interaction in MPT synthase is complex formation is accompanied by a in MoaE as by the (Fig. and for MoaD which is to have an C terminus in its The of residues located in the of MoaD and MoaE for the in MPT synthase formation the complex formation in an in which has to be by the to to the of the MPT synthase A of the MPT synthase active in the form, the orthorhombic form, and the MoaE structure conformational changes the pocket that to be of for the and The pocket consists of the conserved residues from the proximal MoaE subunit and and from the distal MoaE subunit. three and two has an The of is by its in with the of present in all three In a with in the two MPT synthase The pterin moiety of the and product into the binding pocket (Fig. that the in the orthorhombic crystal form A structural of the of the Moco to enzymes that one of the of the cofactor present in Rajagopalan K.V. Schindelin H. J. Chem. Soc. 2000; Scopus Google Scholar) be into the MPT synthase structure that its pterin moiety is in the binding pocket and its terminal phosphate group is in the binding site. conformational changes the two bonds the phosphate group to the be to an This to the MPT complex formation of the dithiolene The pterin system of the is to be in a but to the of the phosphate a system in precursor between MPT synthase and the phosphate group of precursor Z are The of the site in the three MoaE that MPT synthase can precursor Z conformational changes the of and The of the to the is in to the in the position of the between precursor Z and the in the MoaE the one required for precursor sequence of MoaE subunits from that three and are conserved residues and residues and are located in the and their to be the of the The of the residues and be to interact with the phosphate present in the precursor Z or the terminal phosphate present in the product molybdopterin as This two and as residues. and form bonds and interaction with the MoaD C the of these not conservation of these residues as be to form residues are not required to the MoaD C terminus as can be from the MoaD-MoeB where the MoaD C terminus bonds to atoms of MoeB Wuebbens M.M. Rajagopalan K.V. Schindelin H. 2001; PubMed Scopus Google Scholar). The sequence conservation of and that both of these residues in the of MPT The sequence conservation additional conserved residues that be involved in binding the pterin moiety of the In with this is the of the pterin binding site to the in and where atoms are present as binding for the atoms in the pterin Although is a large group of MoaE C-terminal that in the position of this with the C-terminal present in E. coli that of this to an its (12Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) that this is not to a in A of by MPT synthase is the of the two are for the of each precursor Z molecule to a MoaD thiocarboxylate can one sulfur atom. The that the two MoaD C-terminal in heterotetrameric MPT synthase on a precursor Z molecule is by the structure of MPT synthase, the two active are by The that the of and be located in close proximity of the or atoms in precursor Z which the dithiolene moiety of MPT be and to of the MoaD C-terminal the sulfur donor during the that and are one that be either involved in or two in which residues the of these formation of the dithiolene group be a process in which two sulfur atoms are into precursor Z (Fig. The accompanying (12Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) provides for the formation of a that contains a sulfur atom. of the sulfur into precursor Z a of the thiocarboxylate sulfur an be which contains a at either the or two for the are (see Fig. the accompanying (12Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (93) Google the enzyme, its with the either the or of the sulfur into the with of the present in precursor Z more as in the accompanying (12Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (93) Google is for the step during MPT biosynthesis from precursor its with the formation of the containing a sulfur atom. Z must bind in an that to in the incorporation of the sulfur atom. of the sulfur to of the of precursor the terminal phosphate group present in the sulfur is at the these two steps be incorporation at the an to the of precursor The structural from the formation of the terminal group the of the MoaD subunit in present between the MoaD C terminus and the and a binding of the with precursor The binding of the incorporation of the second sulfur at the by binding of a MoaD subunit in the subsequent of binding of the to MPT synthase that it at the active site the MoaD subunits have from the MPT synthase The MoaE homodimer a during the MPT and the in of residues in the the active site that these residues the and it from incorporation of the second sulfur the product be by MPT synthase to to the enzymes the incorporation step during Moco biosynthesis. The structural of MPT synthase in has identified residues involved in binding of the phosphate group present in precursor Z and MPT and revealed conformational changes in two of these residues that binding of the the and the The of MPT synthase has been from the location of the pocket and the MoaD C terminus and a of MPT these that the conserved residues and are involved in the of MPT synthase as in the accompanying (12Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). We the at and of the for