Publications

• GTP cyclohydrolase II structure and mechanism.

  Ren J., Kotaka M., Lockyer M., Lamb H.K., Hawkins A.R., Stammers D.K.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 280:36912-36919(2005) [PubMed] [EuropePMC]

• A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis.

  Frelin O., Huang L., Hasnain G., Jeffryes J.G., Ziemak M.J., Rocca J.R., Wang B., Rice J., Roje S., Yurgel S.N., Gregory J.F. III, Edison A.S., Henry C.S., de Crecy-Lagard V., Hanson A.D.

  Plants and bacteria synthesize the essential human micronutrient riboflavin (vitamin B2) via the same multi-step pathway. The early intermediates of this pathway are notoriously reactive and may be overproduced in vivo because riboflavin biosynthesis enzymes lack feedback controls. In the present ... >> More

  Plants and bacteria synthesize the essential human micronutrient riboflavin (vitamin B2) via the same multi-step pathway. The early intermediates of this pathway are notoriously reactive and may be overproduced in vivo because riboflavin biosynthesis enzymes lack feedback controls. In the present paper, we demonstrate disposal of riboflavin intermediates by COG3236 (DUF1768), a protein of previously unknown function that is fused to two different riboflavin pathway enzymes in plants and bacteria (RIBR and RibA respectively). We present cheminformatic, biochemical, genetic and genomic evidence to show that: (i) plant and bacterial COG3236 proteins cleave the N-glycosidic bond of the first two intermediates of riboflavin biosynthesis, yielding relatively innocuous products; (ii) certain COG3236 proteins are in a multi-enzyme riboflavin biosynthesis complex that gives them privileged access to riboflavin intermediates; and (iii) COG3236 action in Arabidopsis thaliana and Escherichia coli helps maintain flavin levels. COG3236 proteins thus illustrate two emerging principles in chemical biology: directed overflow metabolism, in which excess flux is diverted out of a pathway, and the pre-emption of damage from reactive metabolites. << Less

  Biochem. J. 466:137-145(2015) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli.

  Foor F., Brown G.M.

  An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B. This enzyme is named GTP cyclohydrolase II to ... >> More

  An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B. This enzyme is named GTP cyclohydrolase II to distinguish it from a previously studied E. coli enzyme, named GTP cyclohydrolase (and called GTP cyclohydrolase I in this paper), that catalyzes the first of a series of enzymatic reactions leading to the biosynthesis of the pteridine portion of folic acid (Burg, A. W., and Brown, G. M. (1968) J. Biol. Chem. 243, 2349-2358). Some of the properties of GTP cyclohydrolase II are: (a) divalent cations are required for activity (Mg2+ is most effective); (b) its molecular weight, estimated by filtration on Sephadex G-200, is 44,000; (c) the K-m for GTP is 41 mum; (d) its pH optimum is 8.5; and (e) its activity is inhibited by inorganic pyrophosphate, one of the products of the reaction. Compounds not used as substrate are: GDP, GMP, guanosine, dGTP, ATP, ITP, and XTP. Properties a, b, c, and e (above), as well as the nature of the products, distinguish this enzyme from GTP cyclohydrolase I. Since GTP cyclohydrolase II apparently is not concerned with the biosynthesis of folic acid, the possible physiological role of this enzyme in the biosynthesis of riboflavin is considered in the light of the present investigations and the previously published work on riboflavin biosynthesis by other investigators. << Less

  J. Biol. Chem. 250:3545-3551(1975) [PubMed] [EuropePMC]

• Isolation of cDNAs encoding GTP cyclohydrolase II from Arabidopsis thaliana.

  Kobayashi M., Sugiyama M., Yamamoto K.

  A GTP cyclohydrolase II-encoding gene from Arabidopsis thaliana was isolated through functional complementation of a mutant of Escherichia coli, BSV18, deficient in this protein. The derived amino-acid sequence constitutes a polypeptide of 27 kDa and shows 37-58% identity with previously published ... >> More

  A GTP cyclohydrolase II-encoding gene from Arabidopsis thaliana was isolated through functional complementation of a mutant of Escherichia coli, BSV18, deficient in this protein. The derived amino-acid sequence constitutes a polypeptide of 27 kDa and shows 37-58% identity with previously published sequences of Escherichia coli, Bacillus subtilis, Photobacterium leiognathi and P. phosphoreum. << Less

  Gene 160:303-304(1995) [PubMed] [EuropePMC]

• Biosynthesis of riboflavin: studies on the mechanism of GTP cyclohydrolase II.

  Ritz H., Schramek N., Bracher A., Herz S., Eisenreich W., Richter G., Bacher A.

  GTP cyclohydrolase II catalyzes the first committed reaction in the biosynthesis of the vitamin riboflavin. The recombinant enzyme from Escherichia coli is shown to produce 2,5-diamino-6-beta-ribosylamino-4(3H)-pyrimidinone 5'-phosphate and GMP at an approximate molar ratio of 10:1. The main produ ... >> More

  GTP cyclohydrolase II catalyzes the first committed reaction in the biosynthesis of the vitamin riboflavin. The recombinant enzyme from Escherichia coli is shown to produce 2,5-diamino-6-beta-ribosylamino-4(3H)-pyrimidinone 5'-phosphate and GMP at an approximate molar ratio of 10:1. The main product is subject to spontaneous isomerization affording the alpha-anomer. (18)O from solvent water is incorporated by the enzyme into the phosphate group of the 5-aminopyrimidine derivative as well as GMP. These data are consistent with the transient formation of a covalent phosphoguanosyl derivative of the enzyme. Subsequent ring opening of the covalently bound nucleotide followed by hydrolysis of the phosphodiester bond could then afford the pyrimidine type product. The hydrolysis of the phosphodiester bond without prior ring opening could afford GMP. The enzyme reaction is cooperative with a Hill coefficient of 1.3. Inhibition by pyrophosphate is competitive. Inhibition by orthophosphate is partially uncompetitive at low concentration and competitive at concentrations above 6 mm. << Less

  J. Biol. Chem. 276:22273-22277(2001) [PubMed] [EuropePMC]

• Finding Ways to Relax: A Revisionistic Analysis of the Chemistry of <i>E. coli</i> GTP Cyclohydrolase II.

  Smith M.M., Beaupre B.A., Fourozesh D.C., Meneely K.M., Lamb A.L., Moran G.R.

  Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8-N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α-β phosphodiester bond of the triphosphate to f ... >> More

  Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8-N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α-β phosphodiester bond of the triphosphate to form pyrophosphate (PPi). These hydrolytic reactions are promoted by tandem active-site metal ions, zinc and magnesium, that respectively function at the GTP guanine and triphosphate moieties. The RibA reaction is part of riboflavin biosynthesis and forms 2,5-diamino-6-β-pyrimidinone 5'-phosphate, an exocyclic pyrimidine nucleotide that ultimately forms the pyrimidine ring of the isoalloxazine of riboflavin. The stoichiometry of the RibA reaction was defined in the study that first identified this activity in <i>Escherichia coli</i> (Foor, F., Brown, G. M. <i>J. Biol. Chem.</i>, 1975, 250, 9, 3545-3551) and has not been quantitatively evaluated in subsequent works. Using primarily transient state approaches we examined the interaction of RibA from <i>E. coli</i> with the GTP, inosine triphosphate, and PPi. Our data indicate that PPi is a slow substrate for RibA that is cleaved to form two phosphate ions (Pi). A combination of real-time enzymatically coupled Pi reporter assays and end-point ³¹P NMR revealed that Pi is formed at a catalytically relevant rate in the native reaction of RibA with GTP, redefining the reaction stoichiometry. Furthermore, our data indicate that both PPi and GTP stimulate conformational changes prior to hydrolytic chemistry, and we conclude that the cleavage of PPi bound as a substrate or an intermediate state results in conformational relaxation. << Less

  Biochemistry 60:3027-3039(2021) [PubMed] [EuropePMC]

• Biosynthesis of riboflavin. Single turnover kinetic analysis of GTP cyclohydrolase II.

  Schramek N., Bracher A., Bacher A.

  GTP cyclohydrolase II catalyzes the conversion of GTP into a mixture of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (Compound 2), formate, and pyrophosphate. Moreover, GMP was recently shown to be formed as a minor product. The major product (Compound 2) serves as the first committe ... >> More

  GTP cyclohydrolase II catalyzes the conversion of GTP into a mixture of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (Compound 2), formate, and pyrophosphate. Moreover, GMP was recently shown to be formed as a minor product. The major product (Compound 2) serves as the first committed intermediate in the biosynthesis of the vitamin, riboflavin. Numerous pathogenic microorganisms are absolutely dependent on endogenous synthesis of riboflavin. The enzymes of this pathway are therefore potential drug targets, and mechanistic studies appear relevant for development of bactericidal inhibitors. Pre-steady state quenched flow analysis of GTP cyclohydrolase II shows the rate-determining step to be located at the beginning of the reaction sequence catalyzed by the enzyme. Thus, GTP is consumed at a rate constant of 0.064 s(-1), and the reaction product, Compound 2, is formed at an apparent rate constant of 0.062 s(-1). Stopped flow experiments monitored by multiwavelength photometry are well in line with these data. 2-Amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate can serve as substrate for GTP cyclohydrolase II but does not fulfill the criteria for a kinetically competent intermediate. A hypothetical reaction mechanism involves the slow formation of a phosphoguanosyl derivative of the enzyme under release of pyrophosphate. The covalently bound phosphoguanosyl moiety is proposed to undergo rapid hydrolytic release of formate from the imidazole ring and/or hydrolytic cleavage of the phosphodiester bond. << Less

  J Biol Chem 276:44157-44162(2001) [PubMed] [EuropePMC]

• Arabidopsis RIBA Proteins: two out of three isoforms have lost their bifunctional activity in riboflavin biosynthesis.

  Hiltunen H.M., Illarionov B., Hedtke B., Fischer M., Grimm B.

  Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-p ... >> More

  Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not complemented by the simultaneously expressed isoforms AtRIBA2 and AtRIBA3. The intensity of the bleaching leaf phenotype of RIBA1 deficient plants correlates with the inactivation of AtRIBA1 expression, while no significant effects on the mRNA abundance of AtRIBA2 and AtRIBA3 were observed. We examined reasons why both isoforms fail to sufficiently compensate for a lack of RIBA1 expression. All three RIBA isoforms are shown to be translocated into chloroplasts as GFP fusion proteins. Interestingly, both AtRIBA2 and AtRIBA3 have amino acid exchanges in conserved peptides domains that have been found to be essential for the two enzymatic functions. In vitro activity assays of GCHII and DHBPS with all of the three purified recombinant AtRIBA proteins and complementation of E. coli ribA and ribB mutants lacking DHBPS and GCHII expression, respectively, confirmed the loss of bifunctionality for AtRIBA2 and AtRIBA3. Phylogenetic analyses imply that the monofunctional, bipartite RIBA3 proteins, which have lost DHBPS activity, evolved early in tracheophyte evolution. << Less

  Int. J. Mol. Sci. 13:14086-14105(2012) [PubMed] [EuropePMC]