Histidine 179 Mutants of GTP Cyclohydrolase I Catalyze the Formation of 2-Amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone Triphosphate

Bracher, A.; Fischer, Markus; Eisenreich, Wolfgang; Ritz, Harald; Schramek, Nicholas; Boyle, Peter; Gentili, Patrizia; Huber, Robert; Nar, Herbert; Auerbach, Günter; Bacher, Adelbert

doi:10.1074/jbc.274.24.16727

Cited by 47 publications

(78 citation statements)

References 24 publications

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“…By contrast, the difference spectrum that is observed with SCO 6655 is significantly different (compare difference traces in Figure 3D) from those that are observed with SCO 1441 and SCO 2687. In fact, the UV-visible spectrum of the product of SCO 6655 is similar to that reported for 2-formylamino-5-amino-6-hydroxy-4-(ribosylamino)pyrimidine 5′-phosphate (FAPy), which has an absorbance maximum at 274 nm (13).…”

Section: Spectrophotometric Analysis Of Turnover Of Sco 1441 Sco 268supporting

confidence: 79%

“…In some cases, a [M − + Na + ] complex is also observed. Expanded region of the 2D { 1 H- 13 C} HMBC spectrum of FAPy in 2 H 2 O showing crosspeaks between the formyl proton (H8) and the 13 C resonance at position 5 of the base. Horizontal lines show the 13 C shifts of C6 (base) and C1′ (sugar), where cross-peaks would be expected if the formyl group had been located at N9 instead of N7.…”

Section: Discussionmentioning

confidence: 99%

“…A partial assignment of the 13 C spectrum of FAPy (positions 1′-5′ and 8) has been published (13); the 13 C, 1 H, and 15 N chemical shifts for the product of SCO 6655 are summarized in Table 2-Table 4. The spectra used in the assignments included 1 H and 13 C 1D spectra, 2D { 1 H- 13 (Figures S1 -S5).…”

Section: Analysis Of the Enzymatic Products Of The Three S Coelicolomentioning

confidence: 99%

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Evolution of New Function in the GTP Cyclohydrolase II Proteins of Streptomyces coelicolor

et al. 2006

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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.A substantial role for duplication in evolution of new function was proposed (1-3) long before the recent explosion of genome sequencing and annotation projects revealed the pervasiveness of intragenomic duplications in bacterial genomes. Gene duplication is now widely acknowledged to contribute to evolution of new protein functions by releasing the † This research is supported (in part) by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund (to V.B.).Additional support for V.B. from the NIH (Grant GM 72623) is gratefully acknowledged. J.E.S. is supported by an NIH predoctoral training grant (GM 08804 GCH II belongs to a class of proteins that catalyze cleavage of carbon-8 of GTP to produce structurally related but distinct products (see Figure 1). GCH I catalyzes the conversio...

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Section: Spectrophotometric Analysis Of Turnover Of Sco 1441 Sco 268supporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Analysis Of the Enzymatic Products Of The Three S Coelicolomentioning

confidence: 99%

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Evolution of New Function in the GTP Cyclohydrolase II Proteins of Streptomyces coelicolor

et al. 2006

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“…The apparent K m and K i values for GTP were calculated as described by Cleland (31) to be 46 and 173 M, respectively. The K m value is close to the GTP concentration that gives half-maximal velocity (K 0.5 ) for the rat enzyme (29) and 50-fold higher than the K 0.5 for E. coli GCHI (30). Substrate inhibition has not been reported for other GCHIs.…”

Section: Legchi Does Not Show Cooperative Behavior and Is Inhibited Bmentioning

confidence: 55%

Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I

Basset

Quinlivan

Ziemak

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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GTP cyclohydrolase I (GCHI) mediates the first and committing step of the pterin branch of the folate-synthesis pathway. In microorganisms and mammals, GCHI is a homodecamer of Ϸ26-kDa subunits. Genomic approaches identified tomato and Arabidopsis cDNAs specifying Ϸ50-kDa proteins containing two GCHI-like domains in tandem and indicated that such bimodular proteins occur in other plants. Neither domain of these proteins has a full set of the residues involved in substrate binding and catalysis in other GCHIs. The tomato and Arabidopsis cDNAs nevertheless encode functional enzymes, as shown by complementation of a yeast fol2 mutant and by assaying GCHI activity in extracts of complemented yeast cells. Neither domain expressed separately had GCHI activity. Recombinant tomato GCHI formed dihydroneopterin triphosphate as reaction product, as do other GCHIs, but unlike these enzymes it did not show cooperative behavior and was inhibited by its substrate. Denaturing gel electrophoresis verified that the bimodular GCHI polypeptide is not cleaved in vivo into its component domains, and size-exclusion chromatography indicated that the active enzyme is a dimer. The deduced tomato and Arabidopsis GCHI polypeptides lack overt targeting sequences and thus are presumably cytosolic, in contrast to other plant folate-synthesis enzymes, which are mitochondrial proteins with typical signal peptides. GCHI mRNA and protein are strongly in expressed unripe tomato fruits, implying that fruit folate is made in situ rather than imported. As ripening advances, GCHI expression declines sharply, and folate content drops, suggesting that folate synthesis fails to keep pace with turnover.T etrahydrofolate and its derivatives (folates) are essential cofactors for one-carbon transfer reactions in all organisms. Similar to bacteria and yeasts, plants make folates de novo from pterin, p-aminobenzoate (PABA), and glutamate moieties (1, 2). In contrast, humans and other mammals lack a complete folatesynthesis pathway and thus need dietary folate. Because plant foods are major folate sources, and folate deficiency is a global health problem, enhancing plant folate content is a prime target for metabolic engineering (1, 3). This engineering demands knowledge of the biosynthetic pathway.The plant folate-synthesis pathway is not understood fully, but is most probably similar to that in bacteria (1, 2). The pterin hydroxymethyldihydropteroate is formed from GTP, and PABA from chorismate. The pterin and PABA units are condensed, glutamylated, and reduced to give tetrahydrofolate, and a polyglutamyl tail is added (4). Plant genes and enzymes for the final five steps have been characterized (5, 6), and the enzymes have been shown to be mitochondrial (7,8). Far less is known for plants about the early steps that produce the pterin and PABA moieties.The first step of pterin synthesis is of special interest, because it commits GTP to pterin production and is considered to control flux into the pathway (9, 10). This step, mediated by GTP cyclohydrolase I (GC...

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“…2) (7). GTP cyclohydrolase I was shown to hydrolyze the C-8 -N-9 bond of GTP affording the formamide derivative, Compound 4, which is then converted to the 5-aminopyrimidine derivative, Compound 5, by hydrolytic release of formate (8). Compound 5 subsequently undergoes an Amadori reaction followed by ring closure affording the pteridine chromophore of dihydroneopterin 3Ј-triphosphate (Compound 6).…”

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confidence: 99%

Biosynthesis of Riboflavin

Schramek

Bracher²,

Bacher

2001

Journal of Biological Chemistry

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GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of the vitamin, riboflavin. The enzyme was discovered in Escherichia coli by Foor and Brown (1) who noted the apparently simultaneous formation of three products from GTP, namely formate, pyrophosphate, and an organic compound that was assigned as 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5Ј-phosphate (Compound 2) on basis of indirect evidence (Fig. 1).GTP cyclohydrolase II constitutes a potential antibiotics target for the following reasons: (i) numerous pathogenic bacteria, in particular Gram-negative organisms, are absolutely dependent on endogenous synthesis of the vitamin, because they are unable to absorb the vitamin from the environment because of the absence of an appropriate uptake system; (ii) GTP cyclohydrolase II is absent in animals that are dependent on exogenous supply of vitamin.The exploration of novel antibiotic targets is urgent in light of the fast development of microbial resistance against all antibiotic agents that are currently used. Studies on the reaction mechanism of riboflavin biosynthetic enzymes from bacteria could benefit the development of bactericidal inhibitors. It should also be noted that the enzymes of the riboflavin pathway are potential herbicide targets.The mechanistical details of GTP cyclohydrolase II are incompletely understood. Blau and co-workers (2) reported the formation of two different heterocyclic products but did not determine their structures. The unknown second product was recently identified as the ␣ anomer of Compound 2, which is formed by spontaneous isomerization of the enzyme product and constitutes an in vitro artifact (3). On the other hand, GMP was recently identified as a genuine enzyme product that is formed by GTP cyclohydrolase II at a rate of about 10% as compared with Compound 2 (3).The hydrolytic opening of the imidazole ring of GTP catalyzed by GTP cyclohydrolase II has notable parallels in the reaction catalyzed by GTP cyclohydrolase I, which converts GTP to dihydroneopterin 3Ј-triphosphate, the first committed intermediate in the biosynthetic pathways of tetrahydrobiopterin (4 -6) and tetrahydrofolate ( Fig. 2) (7). GTP cyclohydrolase I was shown to hydrolyze the C-8 -N-9 bond of GTP affording the formamide derivative, Compound 4, which is then converted to the 5-aminopyrimidine derivative, Compound 5, by hydrolytic release of formate (8). Compound 5 subsequently undergoes an Amadori reaction followed by ring closure affording the pteridine chromophore of dihydroneopterin 3Ј-triphosphate (Compound 6). Despite the mechanistic similarity, GTP cyclohydrolases I and II have no detectable sequence similarity.The rate-determining step of GTP cyclohydrolase I occurs relatively late in the reaction catalyzed by GTP cyclohydrolase I (9). In contrast, this paper shows that the rate-determining step of GTP cyclohydrolase II is located at the start of the reaction sequence. -pyrimidinone 5Ј-triphosphate (Compound 4) was prepared as described earlier (8). Recombinant GTP cycl...

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Histidine 179 Mutants of GTP Cyclohydrolase I Catalyze the Formation of 2-Amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone Triphosphate

Cited by 47 publications

References 24 publications

Evolution of New Function in the GTP Cyclohydrolase II Proteins of Streptomyces coelicolor

Evolution of New Function in the GTP Cyclohydrolase II Proteins of Streptomyces coelicolor

Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I

Biosynthesis of Riboflavin

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