Glycerol-3-phosphate Cytidylyltransferase

The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases. Dimeric GCT from Bacillus subtilis displays unusual negative cooperativity in substrate binding and appears to form products only when both active sites are occupied by substrates. Here we describe a complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate. Binding of sulfate to form a pseudo-ternary complex is observed in three of the four chains constituting the asymmetric unit and is accompanied by a backbone rearrangement at Asp11 and ordering of the C-terminal helix. Comparison with the CTP complex of GCT, determined previously, reveals that in the product complex the active site closes around the glycerol phosphate moiety with a concerted motion of the segment 37-47 that includes helix B. This rearrangement allows lysines 44 and 46 to interact with the glycerol and cytosine phosphates of CDP-glycerol. Binding of CDP-glycerol also induces smaller movements of residues 92-100. Roles of lysines 44 and 46 in catalysis have been confirmed by mutagenesis of these residues to alanine, which decreases Vmax(app) and has profound effects on the Km(app) for glycerol-3-phosphate. The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases. Dimeric GCT from Bacillus subtilis displays unusual negative cooperativity in substrate binding and appears to form products only when both active sites are occupied by substrates. Here we describe a complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate. Binding of sulfate to form a pseudo-ternary complex is observed in three of the four chains constituting the asymmetric unit and is accompanied by a backbone rearrangement at Asp11 and ordering of the C-terminal helix. Comparison with the CTP complex of GCT, determined previously, reveals that in the product complex the active site closes around the glycerol phosphate moiety with a concerted motion of the segment 37-47 that includes helix B. This rearrangement allows lysines 44 and 46 to interact with the glycerol and cytosine phosphates of CDP-glycerol. Binding of CDP-glycerol also induces smaller movements of residues 92-100. Roles of lysines 44 and 46 in catalysis have been confirmed by mutagenesis of these residues to alanine, which decreases Vmax(app) and has profound effects on the Km(app) for glycerol-3-phosphate. Cytidylyltransferases function predominantly in biosynthetic pathways where they are responsible for activation of metabolic intermediates. They catalyze reversible reactions in which CTP and an alcohol are substrates, and a cytidylyl ester and pyrophosphate are the products, as shown in Equation 1 (below) for glycerol-3-phosphate cytidylyltransferase (GCT), 1The abbreviations used are: GCTglycerol-3-phosphate cytidylyltransferaseECTethanolamine-phosphate cytidylyltransferaseCCTcholine-phosphate cytidylyltransferasePPATphosphopantetheine adenylyltransferaseNMNATnicotinamide mononucleotide adenylyltransferaseNCSnon-crystallographic symmetry. a bacterial enzyme from Bacillus subtilis. GCT, which functions in the biosynthesis of teichoic acid, a component of certain bacterial cell walls, is a member of a family of cytidylyltransferases that we have been investigating. The other two principal members of this family are phosphocholine and phosphoethanolamine cytidylyltransferases, which function in eukaryotic phospholipid biosynthesis. This family ranges in complexity from multidomain eukaryotic enzymes to simple bacterial prototypes in which the entire protein corresponds to a minimal catalytic domain. The complex cytidylyltransferases have attracted considerable interest, because they are frequently regulatory in biosynthetic pathways, and the activities of some of these enzymes are modulated by membrane lipids (1Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (190) Google Scholar).CTP+glycerol-3-phosphate↔CDP-glycerol+pyrophosphate(Eq. 1) glycerol-3-phosphate cytidylyltransferase ethanolamine-phosphate cytidylyltransferase choline-phosphate cytidylyltransferase phosphopantetheine adenylyltransferase nicotinamide mononucleotide adenylyltransferase non-crystallographic symmetry. We have previously determined the structure of GCT, in complex with its substrate CTP, as a model for the catalytic domain of this family of cytidylyltransferases (2Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold. Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (92) Google Scholar). GCT is a homodimer in which each monomer adopts an α/β fold with a central five-stranded parallel β sheet. In this structure, a large pocket, termed the substrate-binding “bowl,” is formed by helices and loops that extend from the central α/β core (see Fig. 5 in Ref. 2Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold. Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (92) Google Scholar). CTP is bound at one side of the bowl, primarily by interactions with two prominent conserved motifs. The histidine side chains of the 14HWGH motif (3Park Y.-S. Gee P. Sanker S. Schurter E.J. Zuiderweg E.R.P. Kent C. J. Biol. Chem. 1997; 272: 15161-15166Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) interact with the α and β phosphates of the CTP. Both the side chains and backbone atoms of the 113RTEGISTT motif that characterizes this family of cytidylyltransferases (3Park Y.-S. Gee P. Sanker S. Schurter E.J. Zuiderweg E.R.P. Kent C. J. Biol. Chem. 1997; 272: 15161-15166Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) interact with CTP (see below). The GCT structure is closely related to the structures of phosphopantetheine adenylyltransferase (PPAT) (4Izard T. Geerlof A. EMBO J. 1999; 18: 2021-2030Crossref PubMed Scopus (102) Google Scholar, 5Izard T. J. Mol. Biol. 2002; 315: 487-495Crossref PubMed Scopus (65) Google Scholar) and nicotinamide mononucleotide adenylyltransferase (NMNAT) (6D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Structure. 2000; 8: 993-1004Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 7Saradakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 8Zhang H. Zhou T. Kurnasov O. Cheek S. Grishin N.V. Osterman A. Structure. 2002; 10: 69-79Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), key enzymes in cofactor biosyntheses. The GCT, PPAT, and NMNAT structures are representatives of a superfamily of nucleotidyltransferases, all of which are presumed to share a common catalytic fold (9LoConte L. Brenner S.E. Hubbard T. Chothia C. Murzin A.G. Nucleic Acids Res. 2002; 30: 264-267Crossref PubMed Google Scholar, 10Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5594) Google Scholar). They typically incorporate Rossmann-like α/β domains that are topped by a substrate binding region, and they contain the signature HXGH sequence, which forms part of the nucleotide phosphate binding site. The HXGH sequence was first noted in class I aminoacyl-tRNA synthetases (11Borgford T.J. Gray T.E. Brand N.J. Fersht A. Biochemistry. 1987; 26: PubMed Scopus Google Scholar), which are also members of this The GCT family of cytidylyltransferases is from the other in the superfamily by the of the which is to the GCT The structure of the CTP complex (2Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold. Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (92) Google Scholar) interactions of the side of this motif with the substrate also that for the cytosine was primarily by with backbone atoms of the its in the fold is in a of phosphocholine cytidylyltransferase from M. S. Park J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that the structure of this enzyme, which the HXGH and to a family of nucleotidyltransferases, of which catalyze the activation of the binding site for glycerol-3-phosphate and to the that product S. Kent C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar, S. Kent C. Zuiderweg E.R.P. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar), we have determined the structure of GCT with its product, CDP-glycerol (see Fig. The structure is a pseudo-ternary with sulfate as a partial mimic for the second product, and for and in substrate binding and lysines are part of a termed the which closes around the glycerol phosphate of the active site in the product and from was from and enzymes from was from by the of mutagenesis a and with as a to the to the the products of and and the and The for and and for and 1 1 1 The product was the and by the of and in in a with 1 at for and in of 1 and The in a at for 1 the was to a of with The was with GCT was with by the to protein was to by The protein used for was and by on as for GCT in Y.-S. Kent C. J. Biol. Chem. Full Text PDF PubMed Google Scholar) that from was with phosphate of CTP. The protein was by phosphate at was as previously Y.-S. Kent C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). CTP and glycerol-3-phosphate from to that glycerol-3-phosphate was from to for the to the the to and the substrate at was as the of where is the and of and Scholar). from was to by in 1 and 1 CDP-glycerol was to to of 5 to the as for the form of the CTP complex (2Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold. Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (92) Google of the that they with a α β with a of for two asymmetric that as a with an a as the with by The crystal was to and and 1997; 276: Scopus Google Scholar). are in I. The and with a of the observed in this in the with a α β of of in to the in the in to the in the in to the in the in to the in the in a The structure was by the complex as a with the for the GCT (2Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold. Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (92) Google Scholar), CTP, with for and Scholar). The determined the of the first and the The second was from the was in a function for and Scholar). in P. J. M. T. Biol. PubMed Scopus Google Scholar) with and S. A. 1997; PubMed Scopus Google Scholar). with from to used first the model and chains as and was by with non-crystallographic to all this was of and from in bound product and sulfate to the was of and on from A. A. PubMed Scopus Google Scholar), where the chains from one the in In to a core of the structure of the on the of the This to for the four and and to and they and and the of atoms also these to the effects of In the three the to the In these the in and and The for a model with was and was from with from to are in used in and used in of chains and for protein atoms where and are the observed and structure and used in of chains and where and are the observed and structure in a the core that to the of the from and for atoms of residues of chains and The which displays the of the model in J. 26: Google Scholar) that all residues in of the The for the of the product complex are for the other chains and the of this from have been in the with the The chains of I residues and the chains of residues and of the structure of the crystal form of GCT with bound CDP-glycerol was by the structure of the complex as a the asymmetric unit of the crystal includes two I and are four of the fold and of the of the CDP-glycerol with to the for the with of the binding Both chains of I contain with of phosphate to the to the β phosphate of CTP in the substrate complex This bound was as because the in sulfate with The of the sulfate that is a mimic of the other product, pyrophosphate. In this sulfate is in one monomer and in the other of the Binding of CDP-glycerol to of the enzyme for CDP-glycerol to the complex with the Binding to GCT was by the of the of as for the of the S. Kent C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar). in binding of CDP-glycerol as has been observed for CTP and glycerol-3-phosphate. of and for the first and second of product binding to the GCT are to determined for CTP glycerol-3-phosphate S. Kent C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar, S. Kent C. J. Scholar). of GCT with bound product adopts a with the phosphate the and the glycerol moiety the substrate binding helix of the moiety of the product with the 14HWGH and 113RTEGISTT (see and Fig. are to with CTP (2Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold. Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (92) Google Scholar). The side chains of and interact with the sulfate in the that contain shown in the CTP complex (2Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold. Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (92) Google Scholar), of the cytosine moiety to backbone atoms from and the for the cytosine in the CDP-glycerol complex the and interact with the and with the of these have protein in the CTP in product and substrate interactions from a of residues the are of the observed in the two chains of I are of the observed in the two chains of I are of the observed in the two chains of I in a The structure of the complex with CDP-glycerol reveals the binding for glycerol-3-phosphate (see and Fig. The that the and of the product with the of and an of the with The of and the of and form to the the of and both of the of the glycerol moiety (see and Fig. of the of the substrate (see and of the The of the substrate and product in the of GCT are in Fig. The and from CTP and the glycerol phosphate from CDP-glycerol extend on of the The in CDP-glycerol is from that in CTP. two the of this phosphate to and of the in which the a and of the product and CTP are the two structures at residues 37-47 in the of the glycerol phosphate moiety and at residues The the CTP and the product at residues 37-47 is This of the structure, which includes a helix at residues as a that closes the glycerol The concerted rearrangement as a motion and has been the S. Struct. 30: PubMed Scopus Google Scholar, S. Struct. 1999; PubMed Scopus Google Scholar). The motion of the is as a of an the residues and The backbone of in this The smaller at residues and the C-terminal helix are concerted and as In the crystal the two of the asymmetric unit one at an that includes residues from each This the that crystal the of the in the product The chains at this are related by an of and a of and the interactions are from the the substrate of its the sulfate the its this the motion of the is the in all four of the and the of that the of lysines 44 and 46 (see that is and an of crystal of 44 and 46 in of The of the the glycerol-3-phosphate of the product that residues in this for binding the and for The two lysines in this and are conserved in other bacterial glycerol-3-phosphate cytidylyltransferases a conserved is in a segment of eukaryotic phosphocholine and phosphoethanolamine cytidylyltransferases the of and each was to in the of GCT (3Park Y.-S. Gee P. Sanker S. Schurter E.J. Zuiderweg E.R.P. Kent C. J. Biol. Chem. 1997; 272: 15161-15166Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The and as a function of substrate to and substrate Both the and active the enzyme, with the for each enzyme by a of a of cooperativity was was glycerol-3-phosphate. of this the to a This a of the substrate at for substrate at Vmax(app) where the of the for the enzyme to a the was to that was the as substrate at Vmax(app) where the of the for the enzyme to a the was to that was the as The substrate at Vmax(app) where the of S. Kent C. Zuiderweg E.R.P. Struct. Biol. 2001; 8: PubMed Scopus Google the for the enzyme to a the was to that was the as in a The for glycerol-3-phosphate was for each of the This is with the that each a in the In the for CTP for the was for the a that is because all that for one substrate also for the other substrate (3Park Y.-S. Gee P. Sanker S. Schurter E.J. Zuiderweg E.R.P. Kent C. J. Biol. Chem. 1997; 272: 15161-15166Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The and with the sulfate from the appears in three of the four In both chains of I a site to the of CTP, a for one of the phosphates of pyrophosphate (see and Fig. and Fig. in the of the sulfate is bound at a site the of the and of CTP. of is a complex of enzyme and product bound sulfate of this with the chains of I that binding of sulfate to form a pseudo-ternary complex is accompanied by in the nucleotide of the substrate-binding of the chains with and the sulfate is the of the Asp11 and with the of Asp11 the substrate in the complex with sulfate bound and from the in the This also from the in the CTP complex The of this to on the and of the that interact with and In the C-terminal of the is the of the chains in the structure and We this of the has (see of the with the CTP cooperativity S. Kent C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar) one to that substrate binding that to the The to for effects of CTP and CDP-glycerol. of I from the product complex with the CTP complex in the of the monomer chains to one one and by of the interactions as the of of with of are in both CTP and CDP-glycerol are of the to its is by the at Asp11 that in chains binding product and sulfate at the also in the of residues part of a sequence that is conserved members of the GCT family of cytidylyltransferases (2Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold. Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (92) Google Scholar). In the CDP-glycerol complex the side chains of which one at the have that of this side are by CTP binding S. Kent C. Zuiderweg E.R.P. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). In are because of in the and the of the and of the in the of I and the CTP is in the in both the and product to in the interactions of the (see and and the the and the phosphate in the in both other one with the and and two and with in the that with in the CTP complex in the product complex and forms a to the backbone of the a site that also to is to in reactions of GCT Y.-S. Kent C. J. Biol. Chem. Full Text PDF PubMed Google Scholar), in for binding of glycerol-3-phosphate (3Park Y.-S. Gee P. Sanker S. Schurter E.J. Zuiderweg E.R.P. Kent C. J. Biol. Chem. 1997; 272: 15161-15166Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, S. Kent C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar) Sanker and C. and is presumed to the phosphate of and products (6D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Structure. 2000; 8: 993-1004Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, A. H. Struct. Biol. 1997; PubMed Scopus Google Scholar). The that is a in Fig. is in the product complex at a to the of the site observed in the of T. J. Mol. Biol. 2002; 315: 487-495Crossref PubMed Scopus (65) Google Scholar) and in NMNAT from (6D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Structure. 2000; 8: 993-1004Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In these structures is bound to from each of the three phosphates of the which around the with to that this is a binding site for a in the complex of CDP-glycerol with The of the in Binding and with the large family of the HXGH the aminoacyl-tRNA of on a in which catalysis from of the A. and in H. Scholar) by by residues an the catalytic function of the second of the sequence in is by J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of residues has been for the aminoacyl-tRNA synthetases by and Fersht Biochemistry. 1995; PubMed Scopus (65) Google Scholar). of for the residues in the signature of aminoacyl-tRNA synthetases by from to 1995; PubMed Scopus Google Scholar, A. H. G. Biochemistry. PubMed Scopus Google Scholar, Fersht Biochemistry. PubMed Scopus Google Scholar, D. S. G. J. Mol. Biol. PubMed Scopus Google Scholar, T. S. J. Mol. Biol. PubMed Scopus Google Scholar). The signature of aminoacyl-tRNA synthetases corresponds to the of the helix in the structure of GCT (2Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold. Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (92) Google Scholar). The and residues of this sequence interact with CTP and CDP-glycerol the lysines that are for of the in the synthetases are from the GCT signature sequence and are in GCT are conserved and interact with phosphates of the substrate The of the lysines from the motif in aminoacyl-tRNA synthetases to by lysines 44 and 46 from the in of GCT residues to for glycerol-3-phosphate by of an of the of as observed for the lysines in aminoacyl-tRNA The reveals in the of the residues that are presumed to the is in some of the adenylyltransferase enzymes (see which to residues from a sequence for a of these in the of a signature for is to the entire the of residues in the the GCT of cytidylyltransferase as shown in Fig. of for the in this segment of phosphate cytidylyltransferase by a of Kent C. Biochemistry. PubMed Scopus Google Scholar), an for the conserved from the in this member of the GCT have been determined for four with to GCT, from (4Izard T. Geerlof A. EMBO J. 1999; 18: 2021-2030Crossref PubMed Scopus (102) Google Scholar, 5Izard T. J. Mol. Biol. 2002; 315: 487-495Crossref PubMed Scopus (65) Google Scholar) and from and two (6D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Structure. 2000; 8: 993-1004Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 7Saradakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 8Zhang H. Zhou T. Kurnasov O. Cheek S. Grishin N.V. Osterman A. Structure. 2002; 10: 69-79Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). in GCT, the of NMNAT and from incorporate residues that The effects of in GCT that these residues in NMNAT and are to a catalytic shown in Fig. the of the NMNAT enzyme to when and to the phosphates H. Zhou T. Kurnasov O. Cheek S. Grishin N.V. Osterman A. Structure. 2002; 10: 69-79Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). smaller of the is observed in of and (4Izard T. Geerlof A. EMBO J. 1999; 18: 2021-2030Crossref PubMed Scopus (102) Google Scholar). In in the NMNAT enzymes the segment to the of GCT is and interactions with the product phosphates are by side chains at to and of GCT, by residues in the GCT its CTP and glycerol-3-phosphate in Y.-S. Kent C. J. Biol. Chem. Full Text PDF PubMed Google Scholar), displays an form of negative cooperativity in substrate binding S. Kent C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar). the first in binding of the is at the second to the substrate for the CDP-glycerol, displays negative cooperativity in and have that the enzyme only when both active sites are occupied by S. Kent C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar). has also been for PPAT, where structures for (4Izard T. Geerlof A. EMBO J. 1999; 18: 2021-2030Crossref PubMed Scopus (102) Google Scholar, 5Izard T. J. Mol. Biol. 2002; 315: 487-495Crossref PubMed Scopus (65) Google Scholar). cooperativity a of the of the of a GCT from has to for in that of GCT I. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The for negative cooperativity in B. subtilis GCT and for the that the enzyme to are of CTP binding to GCT a model for negative cooperativity S. Kent C. Zuiderweg E.R.P. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). of and backbone as a function of CTP in that to binding of the second CTP and with the residues that for the and also in the C-terminal helix and the conserved The enzyme with and to with cooperativity in binding of CTP, the structures only of the and CDP-glycerol the structures of these two some in the that are with the observed in the appears and adopts are a of the that was in the from the structures that Asp11 and the of in the site. of the structures of substrate and product the at one active site are to the second of the Both the and and in In the of the of the where is in the pyrophosphate with the of the of GCT by S. Kent C. Zuiderweg E.R.P. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). of by that substrate binding in the CTP is with an in the ordering of the chains and the and a for in the of this The of from the with substrate binding to the of negative cooperativity in GCT S. Kent C. Zuiderweg E.R.P. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). of a of GCT is that only when both are bound to both chains S. Kent C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar). of the with the chains of some that are with of the pseudo-ternary from the complex with CDP-glycerol the that in of GCT S. Kent C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar) are in the with CDP-glycerol and to occupied by GCT with glycerol-3-phosphate and the nucleotide was by J. and G. L. the by A. Chem. Scopus Google Scholar). in which the is by an A. Chem. Scopus Google Scholar), have with of the glycerol-3-phosphate site are and are by with of the considerable has been been to of of T. J. Mol. Biol. 2002; 315: 487-495Crossref PubMed Scopus (65) Google Scholar). that binding of and the pyrophosphate to form the complex for the with we in of the and the of in the enzymes related to GCT is that interactions a in both substrate binding and of A. and in H. Scholar, Fersht Biochemistry. 1995; PubMed Scopus (65) Google Scholar). We are to Zuiderweg and for the structure and of