The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.

No abstract

An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184 997-186 043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 lmolAEmg . NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of (S)-a-hydroxyacid oxidizing enzymes including flavocytochrome b 2 and L-lactate dehydrogenase and was shown to catalyse the formation of [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]lactate from [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]pyruvate, albeit at a low rate of 1 nmolAEmg. Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy.

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.GTP cyclohydrolase I catalyzes the formation of dihydroneopterin triphosphate from GTP via a mechanistically complex ring expansion. In plants and micro-organisms, the enzyme product serves as the first committed intermediate in the biosynthesis of tetrahydrofolate (1). In animals, the enzyme product is converted to tetrahydrobiopterin, which serves as cofactor for the biosynthesis of catecholamines and of nitric oxide (2-4). Genetic defects of GTP cyclohydrolase I result in severe neurological impairment (5-8).GTP cyclohydrolase I of Escherichia coli is a 247-kDa homodecamer (9, 10). The structure of the protein has been studied by x-ray structure analysis at a resolution of 2.6 Å (11). The torus-shaped protein obeys D 5 symmetry. Each of the 10 equivalent active sites is located at the interface of three adjacent subunits.Brown, Shiota, and their co-workers (12-14) could show the reaction sequence catalyzed by GTP cyclohydrolase I to involve the opening of the imidazole ring of GTP (compound 1, Fig. 1) with release of formate. Carbon atoms 1Ј and 2Ј of the ribose moiety of GTP are then used to form the dihydropyrazine ring of dihydroneopterin triphosphate (compound 5, Fig. 1). However, the mechanistic details of the highly complex enzymecatalyzed reactions are incompletely understood.The catalytic activity of GTP cyclohydrolase is highly sensitive to the replacement of amino acid residues at the active site cavity (11 13 C]formate, phosphoryl chloride, trimethyl phosphate, N,N-dimethylformamide, tri-n-butylamine, and pyrophosphoric acid were purchased from Sigma-Aldrich. All other chemicals were reagent grade.Enzyme Assays-Assay mixtures contained 100 mM Tris hydrochloride, pH 8.5, 100 mM KCl, 2.5 mM EDTA, 1 mM GTP, and protein in a total volume of 450 l. The mixtures were incubated at 37°C, and 100-l aliquots were retrieved at intervals. The formation of dihydroneopterin triphosphate and 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5Ј-phosphate was monitored as follows.Assay of Dihydroneopterin Triphosphate-Aliquots of enzyme assay mixture were mixed with 30 l of a solution containing 1% iodine and 2% KI in 1 M HCl. After incubation for 30 min at ambient temp...

No abstract

[Structure: see text]. The IspG protein is known to catalyze the transformation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in the nonmevalonate pathway of isoprenoid biosynthesis. We have found that the apparent IspG activity in the cell extracts of recombinant Escherichia coli cells as observed by a radiochemical assay can be enhanced severalfold by coexpression of the isc operon which is involved in the assembly of iron-sulfur clusters. The recombinant protein was isolated by affinity chromatography under anaerobic conditions. With a mixture of flavodoxin, flavodoxin reductase, and NADPH as the reducing agent, stringent assay methods based on photometry or on 13C NMR detection of multiply 13C-labeled substrate/product ratios afforded catalytic activities greater than 60 nmol mg(-1) min(-1) for the protein "as isolated" (i.e., without reconstitution of any kind). Lower apparent activities were found using photoreduced deazaflavin as an artifactual electron donor, whereas dithionite was unable to serve as an artificial electron donor. The apparent Michaelis constant for 2-C-methyl-D-erythritol 2,4-cyclodiphosphate was 700 microM. The enzyme was inactivated by EDTA and could be reactivated by Mn2+. The pH optimum was at 9.0. The protein contained 2.4 iron ions and 4.4 sulfide ions per subunit. The replacement of any of the three conserved cysteine residues afforded mutant proteins which were devoid of catalytic activity and contained less than 6% of Fe2+ and less than 23% of S2- as compared to the wild-type protein. Sequence comparison indicates that putative IspG proteins of plants, the apicomplexan protozoan Plasmodium falciparum, and bacteria from the Bacteroidetes/Chlorobi group contain an insert of about 170-320 amino acid residues as compared with eubacterial enzymes.

GTP cyclohydrolase I catalyzes a ring expansion affording dihydroneopterin triphosphate from GTP. [1,2,3,4,5-13 C 5 ,2-2 H 1 ]GTP was prepared enzymatically from [U-13 C 6 ]glucose for use as enzyme substrate. Multinuclear NMR experiments showed that the reaction catalyzed by GTP cyclohydrolase I involves the release of a proton from C-2 of GTP that is exchanged with the bulk solvent. Subsequently, a proton is reintroduced stereospecifically from the bulk solvent. This is in line with an Amadori rearrangement mechanism. The proton introduced from solvent occupies the pro-7R position in the enzyme product. The data also confirm that the reaction catalyzed by pyruvoyltetrahydropterin synthase results in the incorporation of solvent protons into positions C-6 and C-3 of the enzyme product. On the other hand, the reaction catalyzed by sepiapterin reductase does not involve any detectable incorporation of solvent protons into tetrahydrobiopterin.Pteridines serve as cofactors for a variety of enzyme-catalyzed reactions. Specifically, tetrahydrofolate (in bacteria and eukaryotic organisms) and tetrahydromethanopterin (in archaea) mediate the transfer of one-carbon fragments, tetrahydrobiopterin (BH 4 ) 1 is implicated in the hydroxylation of aromatic amino acids and the formation of nitric oxide in animals (1, 2), and molybdopterin is required as cofactor by a variety of redox enzymes, e.g. xanthine dehydrogenase. The metabolic roles of these cofactors have been reviewed repeatedly (3-6).The formation of pterins by ring expansion of guanosine, including an Amadori rearrangement of the ribose moiety, was first suggested by Weygand et al. (7) on basis of in vivo studies using 14 C-labeled precursors. Subsequent studies by Brown and Burg (8) and by Shiota et al. (9) showed that the first committed step in the biosynthesis of tetrahydrofolate and BH 4 is catalyzed by the enzyme GTP cyclohydrolase I. More specifically, C-8 of GTP (Fig. 1, compound 1) is released as formate, carbon atoms 1Ј and 2Ј of the ribose moiety are utilized for the formation of the dihydropyrazine ring, and carbon atoms 3Ј-5Ј of GTP afford the position 6 side chain of dihydroneopterin triphosphate (NH 2 TP) (Fig. 1, compound 2) (for a review, see Ref.3).The product of GTP cyclohydrolase I, NH 2 TP, is converted to BH 4 (compound 4) by the consecutive action of pyruvoyltetrahydropterin synthase (PPH 4 synthase) and sepiapterin reductase (10 -12). PPH 4 synthase catalyzes the elimination of triphosphate from NH 2 TP as well as a series of tautomerization reactions that are conducive to the formation of a tetrahydropterin from the dihydropterin substrate. Both carbonyl groups of the resulting pyruvoyltetrahydropterin (PPH 4 , compound 3) are subsequently reduced by the action of sepiapterin reductase.The three-dimensional structures of GTP cyclohydrolase I from Escherichia coli (13, 14), PPH 4 synthase from rat (15, 16), and sepiapterin reductase (17) from mouse have been determined by x-ray crystallography. The folding patterns of GTP cyclohydrolase I ...

No abstract