mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites.

Characterization of the donor and acceptor specificities of mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase isolated from vaccinia virus cores has enabled us to discriminate between alternative reaction sequences leading to the formation of the 5'-terminal m7G(5')pppN-structure. The mRNA guanylyltransferase catalyzes the transfer of a residue of GMP from GTP to acceptors which possess a 5'-terminal diphosphate. A diphosphate-terminated polyribonucleotide is preferred to a mononucleoside diphosphate as an acceptor suggesting that the guanylyltransferase reaction occurs after initiation of RNA synthesis. Although all of the homopolyribonucleotides tested (pp(A)n, pp(G)n, pp(I)n, pp(U)n, and pp(C)n) are acceptors for the mRNA guanylyltransferase indicating lack of strict sequence specificity, those containing purines are preferred. Only GTP and dGTP are donors in the reaction; 7-methylguanosine (m7G) triphosphate specifically is not a donor indicating that guanylylation must precede guanine-7-methylation. The preferred acceptor of the mRNA (guanine-7-)-methyltransferase is the product of the guanylyltransferase reaction, a polyribonucleotide with the 5'-terminal sequence G(5')pppN-. The enzyme can also catalyze, but less efficiently methylation of the following: dinucleoside triphosphates with the structure G(5')pppN, GTP, dGTP, ITP, GDP, GMP, and guanosine. The enzyme will not catalyze the transfer of methyl groups to ATP, XTP, CTP, UTP, or to guanosine-containing compounds with phosphate groups in either positions 2' or 3' or in 3'-5' phosphodiester linkages. The latter specificity provides an explanation for the absence of internal 7-methylguanosine in mRNA. In the presence of PPi, the mRNA guanylyltransferase catalyzes the pyrophosphorolysis of the dinucleoside triphosphate G(5')pppA, but not of m7G(5')pppA. Since PPi is generated in the process of RNA chain elongation, stabilization of the 5'-terminal sequences of mRNA is afforded by guanine-7-methylation.     

Analysis of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) enzyme of Salmonella typhimurium LT2. Identification of residue His-46 as the site of guanylylation

CobU is a bifunctional enzyme involved in adenosylcobalamin (coenzyme B(12)) biosynthesis in Salmonella typhimurium LT2. In this bacterium, CobU is the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase needed to convert cobinamide to adenosylcobinamide-GDP during the late steps of adenosylcobalamin biosynthesis. The guanylyltransferase reaction has been proposed to proceed via a covalently modified CobU-GMP intermediate. Here we show that CobU requires a nucleoside upper ligand on cobinamide for substrate recognition, with the nucleoside base, but not the 2'-OH group of the ribose, being important for this recognition. During the kinase reaction, both the nucleotide base and the 2'-OH group of the ribose are important for gamma-phosphate donor recognition, and GTP is the only nucleotide competent for the complete nucleotidyltransferase reaction. Analysis of the ATP:adenosylcobinamide kinase reaction shows CobU becomes less active during this reaction due to the formation of a covalent CobU-AMP complex that holds CobU in an altered conformation. Characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase reaction shows the covalent CobU-GMP intermediate is on the reaction pathway for the generation of adenosylcobinamide-GDP. Identification of a modified histidine and analysis of cobU mutants indicate that histidine 46 is the site of guanylylation.     

Purification and characterization of the messenger ribonucleic acid capping enzyme GTP:RNA guanylyltransferase from wheat germ

A GTP:RNA guanylyltransferase or capping enzyme has been purified approximately 2000-fold from wheat germ. The enzyme catalyzes the transfer of the GMP residue from GTP to the 5' end of RNA or synthetic polyribonucleotides. Diphosphate-ended polymers were capped more efficiently than molecules with triphosphate ends, and molecules with monophosphate ends were not capped at all. There appears to be little specificity since RNAs with purine or pyrimidine ends served as acceptors. Other features of the wheat germ RNA guanylyltransferase include relatively low Km values for GTP (2.7 microM) and ppA (pA)n (14.2 nM), a divalent cation requirement satisfied by low (0.5 mM) concentrations of MnCl2 or higher (5 mM) concentrations of MgCl2, and a pH optimum around neutrality.     

Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) complexed with GMP: evidence for a substrate-induced transferase active site.

The X-ray crystal structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) from Salmonella typhimurium bound to GMP has been determined by molecular replacement to 2.2 A resolution. CobU is a bifunctional enzyme, which catalyzes the phosphorylation of the 1-amino-O-2-propanol side chain of the adenosylcobinamide ring and subsequently functions as a guanylyltransferase to form adenosylcobinamide.GDP. The transferase activity involves a covalent enzyme-guanylyl intermediate that is most likely a phosphoramidate linkage to His(46). Previous studies have shown that the enzyme is a homotrimer and adopts a pinwheel shape. Each subunit consists of a single domain of six parallel beta-strands and one antiparallel strand flanked on either side by a total of five alpha-helices and one helical turn. Interestingly, His(46) in the apoenzyme is located a considerable distance from the kinase active site or P-loop motif and is solvent-exposed [Thompson, T. B., et al. (1998) Biochemistry 37, 7686-7695]. To examine the structural relationship of the two active sites, CobU was cocrystallized with GTP and pyrophosphate. Crystals belong to space group P2(1)2(1)2(1) with the following unit cell dimensions: a = 58. 4 A, b = 87.8 A, and c = 101.6 A. The structure shows electron density for the hydrolysis product GMP rather than the expected covalent guanylyl intermediate which appears to have been hydrolyzed in the crystal lattice. Even so, CobU exhibits a substantial conformational rearrangement. The helix axis containing His(46), the site of guanylylation, rotates 30 degrees and translates 11 A relative to the apo structure and is accompanied by compensatory unwinding and rewinding at the helix ends to allow the induction of a guanosine binding pocket between beta-strand 2 and alpha-helix 2. This conformational change brings the C(alpha) of His(46) approximately 10 A closer to the P-loop motif such that a phosphate ion located in the P-loop is only 6 A from the alpha-phosphate of GMP. This suggests that the P-loop motif may be used to coordinate the terminal phosphates in both the transferase and kinase reactions and implies that the active sites for both reactions overlap.     

A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities.

The two consecutive activities of the cobalamin biosynthetic pathway that catalyze the conversion of cobinamide to cobinamide phosphate (cobinamide kinase) and of cobinamide phosphate to GDP-cobinamide (cobinamide phosphate guanylytransferase) were shown to be carried by the same protein in Pseudomonas denitrificans. This bifunctional protein was purified to homogeneity by high-performance liquid chromatography of extracts of a recombinant strain of this microorganism, and the sequence of the first 10 amino acid residues at the N terminus was determined. Both activities were specific to the coenzyme forms of the corrinoid substrates and exhibited an optimum pH at 8.8. Both ATP and GTP were shown to be in vitro gamma-phosphate donors for cobinamide kinase. However, competition experiments demonstrated that ATP was the preferred substrate, a result that can be explained in terms of the kinetic properties of the enzyme. Labeling experiments established that the phosphate group of cobinamide phosphate is quantitatively retained as the inner phosphate of GDP-cobinamide during the guanylyltransferase reaction. The native protein had an apparent molecular weight of 40,000, as estimated by gel filtration, and consisted of two identical subunits of Mr 20,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein had an isoelectric point of 5.35 and contained a high-affinity GTP-binding site (Kaff.(GTP) = 0.22 microM). Binding of GTP onto this site resulted in a marked increase of the affinity of cobinamide kinase for cobinamide. This property and other kinetic properties may regulate the enzyme and prevent the accumulation of cobinamide phosphate.     

Purification of mRNA guanylyltransferase from calf thymus.

mRNA guanylyltransferase has been extensively purified from calf thymus. A GTP-binding assay was used based on the observations by Shuman and Hurwitz (1981) and Venkatesan and Moss (1982) that vaccinia virus and HeLa cell mRNA guanylyltransferases bind the GMP moiety from GTP in the absence of an acceptor RNA. The mol. wt. of the purified enzyme from calf thymus, estimated by polyacrylamide gel electrophoresis in the presence of SDS, is 65 000. The major protein in the purified enzyme fraction comigrates with the peptide labelled with GMP. Based on scans of silver-stained polyacrylamide gels, mRNA guanylyltransferase constitutes greater than 50% of the protein in these fractions. The enzyme catalyzed the guanylylation at the 5' end of poly(A) with a mixture of diphosphate and triphosphate ends. No evidence was obtained for a direct interaction between mRNA guanylyltransferase and RNA polymerase B (II).     

Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme)

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.     

Assembly and protection of the radical enzyme, methylmalonyl-CoA mutase, by its chaperone

MeaB is a recently described P-loop GTPase that plays an auxiliary role in the reaction catalyzed by the radical B12 enzyme, methylmalonyl-CoA mutase. Defects in the human homologue of MeaB result in methylmalonic aciduria, but the role of this protein in coenzyme B12 assimilation and/or utilization is not known. Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA that uses reactive radical intermediates that are susceptible to oxidative inactivation. In this study, we have examined the influence of MeaB on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase and on the thermodynamics of cofactor binding. MeaB alone has a modest effect on the affinity of the mutase for the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor, increasing it 2-fold from 404 +/- 71 to 210 +/- 22 nM. However, in the presence of GDP, the affinity for the cofactor decreases 5-fold to 1.89 +/- 0.33 microM, while in the presence of guanosine 5'(beta-gamma imino)triphosphate, a nonhydrolyzable analogue of GTP, the binding of AdoCbl to the mutase is not detected. Protection against oxidative inactivation of the mutase by MeaB is dependent upon the presence of nucleotides with the MeaB/GDP and MeaB/GTP complexes decelerating the rate of formation of oxidized cofactor by 3- and 15-fold, respectively. This study suggests that MeaB functions in the GTP-dependent assembly of holomethylmalonyl-CoA mutase and subsequent protection of radical intermediates during catalysis.     

Mutational analysis of the guanylyltransferase component of Mammalian mRNA capping enzyme

RNA guanylyltransferase is an essential enzyme that catalyzes the second of three steps in the synthesis of the 5'-cap structure of eukaryotic mRNA. Here we conducted a mutational analysis of the guanylyltransferase domain of the mouse capping enzyme Mce1. We introduced 50 different mutations at 22 individual amino acids and assessed their effects on Mce1 function in vivo in yeast. We identified 16 amino acids as being essential for Mce1 activity (Arg299, Arg315, Asp343, Glu345, Tyr362, Asp363, Arg380, Asp438, Gly439, Lys458, Lys460, Asp468, Arg530, Asp532, Lys533, and Asn537) and clarified structure-activity relationships by testing the effects of conservative substitutions. The new mutational data for Mce1, together with prior mutational studies of Saccharomyces cerevisiae guanylyltransferase and the crystal structures of Chlorella virus and Candida albicans guanylyltransferases, provide a coherent picture of the functional groups that comprise and stabilize the active site. Our results extend and consolidate the hypothesis of a shared structural basis for catalysis by RNA capping enzymes, DNA ligases, and RNA ligases, which comprise a superfamily of covalent nucleotidyl transferases defined by a constellation of conserved motifs. Analysis of the effects of motif VI mutations on Mce1 guanylyltransferase activity in vitro highlights essential roles for Arg530, Asp532, Lys533, and Asn537 in GTP binding and nucleotidyl transfer.     

GTP cyclohydrolase II structure and mechanism

GTP cyclohydrolase II converts GTP to 2,5-diamino-6-beta-ribosyl-4(3H)-pyrimidinone 5'-phosphate, formate and pyrophosphate, the first step in riboflavin biosynthesis. The essential role of riboflavin in metabolism and the absence of GTP cyclohydrolase II in higher eukaryotes makes it a potential novel selective antimicrobial drug target. GTP cyclohydrolase II catalyzes a distinctive overall reaction from GTP cyclohydrolase I; the latter converts GTP to dihydroneopterin triphosphate, utilized in folate and tetrahydrobiopterin biosynthesis. The structure of GTP cyclohydrolase II determined at 1.54-A resolution reveals both a different protein fold to GTP cyclohydrolase I and distinctive molecular recognition determinants for GTP; although in both enzymes there is a bound catalytic zinc. The GTP cyclohydrolase II.GMPCPP complex structure shows Arg(128) interacting with the alpha-phosphonate, and thus in the case of GTP, Arg(128) is positioned to act as the nucleophile for pyrophosphate release and formation of the proposed covalent guanylyl-GTP cyclohydrolase II intermediate. Tyr(105) is identified as playing a key role in GTP ring opening; it is hydrogen-bonded to the zinc-activated water molecule, the latter being positioned for nucleophilic attack on the guanine C-8 atom. Although GTP cyclohydrolase I and GTP cyclohydrolase II both use a zinc ion for the GTP ring opening and formate release, different residues are utilized in each case to catalyze this reaction step.     

Hydrolysis of GTP by the alpha-chain of Gs and other GTP binding proteins

The functions of G proteins--like those of bacterial elongation factor (EF) Tu and the 21 kDa ras proteins (p21ras)--depend upon their abilities to bind and hydrolyze GTP and to assume different conformations in GTP- and GDP-bound states. Similarities in function and amino acid sequence indicate that EF-Tu, p21ras, and G protein alpha-chains evolved from a primordial GTP-binding protein. Proteins in all three families appear to share common mechanisms for GTP-dependent conformational change and hydrolysis of bound GTP. Biochemical and molecular genetic studies of the alpha-chain of Gs (alpha s) point to key regions that are involved in GTP-dependent conformational change and in hydrolysis of GTP. Tumorigenic mutations of alpha s in human pituitary tumors inhibit the protein's GTPase activity and cause constitutive elevation of adenylyl cyclase activity. One such mutation replaces a Gln residue in alpha s that corresponds to Gln-61 of p21ras; mutational replacements of this residue in both proteins inhibit their GTPase activities. A second class of GTPase inhibiting mutations in alpha s occurs in the codon for an Arg residue whose covalent modification by cholera toxin also inhibits GTP hydrolysis by alpha s. This Arg residue is located in a domain of alpha s not represented in EF-Tu or p21ras. We propose that this domain constitutes an intrinsic activator of GTP hydrolysis, and that it performs a function analogous to that performed for EF-Tu by the programmed ribosome and for p21ras by the recently discovered GTPase-activating protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

GTP is required to stabilize and display transamidation activity of transglutaminase 2

Transglutaminase 2 (TGase 2) is a bifunctional enzyme that catalyzes calcium-dependent transamidation and GTP binding/hydrolysis. The transamidation activity is proposed to be associated with several neurodegenerative disorders such as Alzheimer's and Hungtinton's disease. However, the regulation mechanism by which TGase 2 causes neurodegeneration is unknown. In this study, we show that two activities of TGase 2 have a differential stability; transamidation activity is less stable than GTP hydrolytic activity, and that GTP was required to stabilize and to display transamidation activity. Moreover, GTP binding-defective mutant of TGase 2 did not show any transamidation activity in transfection experiments. These results indicate that GTP binding is crucial for transamidation activity of TGase 2, suggesting that protein cross-linking by TGase 2 might be associated with G-protein coupled receptor signaling system. Thus, our data could contribute to understand the regulation of TGase 2 activity and TGase 2-associated pathogenesis.     

Novel coenzyme B12-dependent interconversion of isovaleryl-CoA and pivalyl-CoA

5'-Deoxyadenosylcobalamin (AdoCbl)-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently characterized a fusion protein that comprises the two subunits of the AdoCbl-dependent isobutyryl-CoA mutase flanking a G-protein chaperone and named it isobutyryl-CoA mutase fused (IcmF). IcmF catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA, whereas GTPase activity is associated with its G-protein domain. In this study, we report a novel activity associated with IcmF, i.e. the interconversion of isovaleryl-CoA and pivalyl-CoA. Kinetic characterization of IcmF yielded the following values: a K(m) for isovaleryl-CoA of 62 ± 8 μM and V(max) of 0.021 ± 0.004 μmol min(-1) mg(-1) at 37 °C. Biochemical experiments show that an IcmF in which the base specificity loop motif NKXD is modified to NKXE catalyzes the hydrolysis of both GTP and ATP. IcmF is susceptible to rapid inactivation during turnover, and GTP conferred modest protection during utilization of isovaleryl-CoA as substrate. Interestingly, there was no protection from inactivation when either isobutyryl-CoA or n-butyryl-CoA was used as substrate. Detailed kinetic analysis indicated that inactivation is associated with loss of the 5'-deoxyadenosine moiety from the active site, precluding reformation of AdoCbl at the end of the turnover cycle. Under aerobic conditions, oxidation of the cob(II)alamin radical in the inactive enzyme results in accumulation of aquacobalamin. Because pivalic acid found in sludge can be used as a carbon source by some bacteria and isovaleryl-CoA is an intermediate in leucine catabolism, our discovery of a new isomerase activity associated with IcmF expands its metabolic potential.     

GTP stimulates and inhibits adenylate cyclase in fat cell membranes through distinct regulatory processes.

GTP and hormones activate, synergistically, adenylate cyclase in purified plasma membranes from rat adipocytes. Addition of chelating reagents (EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) or thiol-reducing reagents (dithiothreitol or 2-mercaptoethanol) results in marked inhibition of enzyme activity without altering the synergistic stimulatory effects of GTP and hormones. The inhibitory effects of the reagents required the presence of GTP, indicating that inhibition involves a GTP-dependent process. This process is separate from the GTP-dependent process responsible for activation of the enzyme since it is selectively abolished by pretreatment of fat cell membranes with trypsin. It is suggested that inhibition and activation of fat cell adenylate cyclase by GTP occur through distinct regulatory processes.     

Histidine 179 mutants of GTP cyclohydrolase I catalyze the formation of 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone triphosphate.

GTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. The replacement of histidine 179 by other amino acids affords mutant enzymes that do not catalyze the formation of dihydroneopterin triphosphate. However, some of these mutant proteins catalyze the conversion of GTP to 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-triphosphate as shown by multinuclear NMR analysis. The equilibrium constant for the reversible conversion of GTP to the ring-opened derivative is approximately 0.1. The wild-type enzyme converts the formylamino pyrimidine derivative to dihydroneopterin triphosphate; the rate is similar to that observed with GTP as substrate. The data support the conclusion that the formylamino pyrimidine derivative is an intermediate in the overall reaction catalyzed by GTP cyclohydrolase I.     

Polyethylene glycol-stimulated microsomal GTP hydrolysis. Relationship to GTP-mediated Ca2+ release

It has recently been observed that GTP mediates Ca2+ release from internal Ca2+ stores. In contrast to effects on permeabilized cells, GTP-dependent Ca2+ release in isolated microsomes requires the presence of polyethylene glycol (PEG). We have investigated the effects of PEG on microsomal GTPase activity and report that PEG stimulates a high-affinity (Km = 0.9 microM) GTPase. The effects of PEG reflect an increase in the Vmax of this activity; no effects on Km were observed. The concentration dependence for PEG-dependent stimulation of the high-affinity GTPase exactly mimicked that for GTP-dependent Ca2+ release. The stimulation of GTP hydrolysis by PEG was specific for the microsome fraction; only small effects were obtained with plasma membrane or cytosol fractions. As observed for GTP-dependent Ca2+ release, the microsomal PEG-stimulated GTPase was competitively inhibited by the GTP analog GTP gamma S (Ki = 60 nM). It is proposed that the PEG-stimulated GTPase may represent an intrinsic activity of the guanine nucleotide binding protein involved in the regulation of reticular Ca2+ fluxes.     

Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae. II. Catalytic mechanism.

Yeast histidine tRNA guanylyltransferase (TGT) catalyzes in the presence of ATP the addition of GTP to the 5' end of eukaryotic cytoplasmic tRNAHis species. A study of the enzyme mechanism with purified protein showed that during the first step ATP is cleaved to AMP and PPi creating adenylylated TGT. In a second step the activated enzyme forms a stable complex with its cognate tRNA substrate. The 5'-phosphate of the tRNA is adenylylated by nucleotide transfer from the adenylylated guanylyltransferase to form A(5')pp(5')N at the 5'-end of the tRNA. Finally, the 3'-hydroxyl of GTP adds to the activated 5' terminus of the tRNA with the release of AMP. This mechanism of tRNAHis guanylyltransferase is very similar to that of RNA ligases. dATP can substitute for ATP in this reaction. Since among several guanosine compounds active in this reaction GTP is most efficiently added we believe that it is the natural substrate of TGT.     

Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii

GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by ¹H-¹⁵N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobY^G153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobY^G153D:GTP complex was also found to be unstable over time.     

Evolution of new function in the GTP cyclohydrolase II proteins of Streptomyces coelicolor

The genome sequence of Streptomyces coelicolor contains three open reading frames (sco1441, sco2687, and sco6655) that encode proteins with significant (>40%) amino acid identity to GTP cyclohydrolase II (GCH II), which catalyzes the committed step in the biosynthesis of riboflavin. The physiological significance of the redundancy of these proteins in S. coelicolor is not known. However, the gene contexts of the three proteins are different, suggesting that they may serve alternate biological niches. Each of the three proteins was overexpressed in Escherichia coli and characterized to determine if their functions are biologically overlapping. As purified, each protein contains 1 molar equiv of zinc/mol of protein and utilizes guanosine 5'-triphosphate (GTP) as substrate. Two of these proteins (SCO 1441 and SCO 2687) produce the canonical product of GCH II, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (APy). Remarkably, however, one of the three proteins (SCO 6655) converts GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), as shown by UV-visible spectrophotometry, mass spectrometry, and NMR. This activity has been reported for a GTP cyclohydrolase III protein from Methanocaldococcus jannaschii [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084], which has no amino acid sequence homology to SCO 6655. Comparison of the sequences of these proteins and mapping onto the structure of the E. coli GCH II protein [Ren, J., Kotaka, M., Lockyer, M., Lamb, H. K., Hawkins, A. R., and Stammers, D. K. (2005) J. Biol. Chem. 280, 36912-36919] allowed identification of a switch residue, Met120, which appears to be responsible for the altered fate of GTP observed with SCO 6655; a Tyr is found in the analogous position of all proteins that have been shown to catalyze the conversion of GTP to APy. The Met120Tyr variant of SCO 6655 acquires the ability to catalyze the conversion of GTP to APy, suggesting a role for Tyr120 in the late phase of the reaction. Our data are consistent with duplication of GCH II in S. coelicolor promoting evolution of a new function. The physiological role(s) of the gene clusters that house GCH II homologues will be discussed.     

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.