Biosynthesis of riboflavin: structure and properties of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase of Methanocaldococcus jannaschii

The pyrimidine reductase of the riboflavin biosynthetic pathway (MjaRED) specified by the open reading frame MJ0671 of Methanocaldococcus jannaschii was expressed in Escherichia coli using a synthetic gene. The synthetic open reading frame that was optimized for expression in E. coli directed the synthesis of abundant amounts of the enzyme with an apparent subunit mass of 25 kDa. The enzyme was purified to apparent homogeneity and was shown to catalyze the conversion of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate at a rate of 0.8 micromol min(-1) mg(-1) at pH 8.0 and at 30 degrees C. The protein is a homodimer as shown by sedimentation equilibrium analysis and sediments at an apparent velocity of 3.5 S. The structure of the enzyme in complex with the cofactor nicotinamide adenine dinucleotide phosphate was determined by X-ray crystallography at a resolution of 2.5 Angstroms. The folding pattern resembles that of dihydrofolate reductase with the Thermotoga maritima ortholog as the most similar structure. The substrate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, was modeled into the putative active site. The model suggests the transfer of the pro-R hydrogen of C-4 of NADPH to C-1' of the substrate.     

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.     

A bioluminescence method for determining adenosine 3'-phosphate 5'-phosphate (PAP) and adenosine 3'-phosphate 5'-sulfatophosphate (PAPS) in biological materials.

A rapid, sensitive, bioluminescence technique for detecting PAPS (adenosine 3′-phosphate 5′-sulfatophosphate) in biological materials is described. PAPS is first hydrolysed in 0.2 n HCl to PAP (adenosine 3′-phosphate 5′-phosphate) and is then assayed by the luciferin-luciferase system of the sea pansy, Renilla reniformis, which is specific for PAP. This bioluminescence system produces light at a rate that is proportional to the amount of PAP present. Light emission is measured in a liquid scintillation spectrometer with the two photomultipliers out of coincidence.Very low amounts of PAPS (10–100 pmoles) have been determined in extracts of yeast and various plant tissues by this method. The production of PAPS in extracts of young wheat leaves is enhanced by including either 5′-AMP or 3′-AMP in the reaction mixture. It is possible that these nucleotides protect PAPS from enzymes that degrade this compound, e.g., a nucleotidase.     

Studies on N4-(2-deoxy-D-pentofuranosyl)-4,6-diamino-5-formamidopyrimidine (Fapy.dA) and N6-(2-deoxy-D-pentofuranosyl)-6-diamino-5-formamido-4-hydroxypyrimidine (Fapy.dG).

Exposure of DNA to oxidative stress produces a variety of DNA lesions including the formamidopyrimidines, which are derived from the purines. These lesions may play important roles in carcinogenesis. We achieved the first chemical syntheses of a monomeric form of Fapy-dA (1) and oligonucleotides containing this lesion or Fapy-dG at a defined site. Monomeric Fapy-dA readily epimerized at 25 degrees C in phosphate buffer (pH 7.5). The beta-anomer was favored by a ratio of 1.33:1.0, and equilibration was achieved in less than 7 h. Deglycosylation of Fapy-dA in the monomer follows first-order kinetics from 37 to 90 degrees C. The rate constants for deglycosylation of Fapy-dA in the monomeric and oligonucleotide substrates were measured at a common temperature (55 degrees C) and found to be the same within experimental error (t(1/2) = 20.5 h). Implementation of the activation parameters measured for the deglycosylation of 1 indicates that the half-life for deglycosylation of Fapy-dA at 37 degrees C is approximately 103 h. Analysis of the rate constant for deglycosylation of Fapy-dG in an oligonucleotide, revealed that this lesion is approximately 25 times more resistant to hydrolysis than Fapy-dA at 55 degrees C. These results indicate that Fapy-dA and Fapy-dG will be sufficiently long-lived in DNA so as to warrant investigation of their genotoxicity, and both anomers will be present during this time.     

NADP(H) phosphatase activities of archaeal inositol monophosphatase and eubacterial 3'-phosphoadenosine 5'-phosphate phosphatase

NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP+ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3'-phosphoadenosine 5'-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3'-phosphoadenosine 5'-phosphate phosphatase. Based on this fact, we found that 3'-phosphoadenosine 5'-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3'-phosphoadenosine 5'-phosphate phosphatase rather than as NADP(H) phosphatase.     

Occurrence and biosynthesis of 5-aminoimidazole-4-carboxamide ribonucleotide and N-(beta-D-ribofuranosyl)formamide 5'-phosphate in Methanobacterium thermoautotrophicum delta(H).

5-Aminoimidazole-4-carboxamide ribonucleotide (ZMP) and N-(beta-D-ribofuranosyl)formamide 5'-phosphate (FAR-P) have been identified as products of the metabolism of ATP and 5-phospho-alpha-D-ribosyl diphosphate by Methanobacterium thermoautotrophicum delta(H), a member of the domain Archaea. Evidence indicates that the first three steps in the pathway to the formation of these compounds are the same as the first three steps of histidine biosynthesis and lead to the generation of pro-phosphoribosyl formimino-5-aminoimidazole-4-carboxamide ribonucleotide (5'-proFAR). The 5'-proFAR then undergoes hydrolysis to ZMP and FAR-P. The reaction was detected by an unexpected high concentration of ZMP in cell extracts of M. thermoautotrophicum delta(H).     

Km and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one with dihydropteridine reductase.

The Km and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one were determined for dihydropteridine reductase (EC 1.6.99.10) from two sources. The parameters of the pterin are of the same order as those of the most effective substrates of dihydropteridine reductase. The Km values of the pterin are one order of magnitude smaller than those of the pyrimidinone, although the kcat. values are of the same order.     

Glucopyranosides derived from 6-aryl-5-cyano-2-(methylthio)pyrimidin-4(3H)ones

The reaction of 2,3.4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide with a 6-aryl-5-cyano-2-(methylthio)pyrimidin-4(3H)one in aqueous acetone in the presence of KOH furnishes a 4-(beta-D-glucopyranosyloxy)pyrimidine and a 3-(beta-D-glucopyranosyl)pyrimidine as the major and minor product. respectively.     

N-(5'-phospho-4'-pyridoxyl)amines as substrates for pyridoxine (pyridoxamine) 5'-phosphate oxidase

A preparation of pyridoxine (pyridoxamine) 5′-phosphate oxidase, with a specific activity of 9,400 nmoles/hr/mg protein, 10-fold higher than that previously reported, was used to study the oxidation of various N-(5′-phospho-4′-pyridoxyl)amines. Values for K_m, from 3.1 × 10^?5 M to 1.6 × 10^?3 M, and for V_max, relative to pyridoxamine-P, of 20 to 140% were obtained. Compounds lacking a 5′-phosphate were not substrates, and the enzymic reaction was dependent on the presence of both FMN and O₂. N-(phosphopyridoxyl)-L-amino acids had lower K_m's than the corresponding -D-amino acid compounds. When 1-¹⁴C-N-(phosphopyridoxyl)glycine was used as a substrate, no ¹⁴CO₂ was evolved, and 1-¹⁴C-glycine was detected in the incubation mixture.     

Transformation of vanadinite [Pb5(VO4)3Cl] by fungi

Saprotrophic fungi were investigated for their bioweathering effects on the vanadium‐ and lead‐containing insoluble apatite group mineral, vanadinite [Pb₅(VO₄)₃Cl]. Despite the insolubility of vanadinite, fungi exerted both biochemical and biophysical effects on the mineral including etching, penetration and formation of new biominerals. Lead oxalate was precipitated by Aspergillus niger during bioleaching of natural and synthetic vanadinite. Some calcium oxalate monohydrate (whewellite) was formed with natural vanadinite because of the presence of associated ankerite [Ca(Fe²⁺,Mg)(CO₃)₂]. Aspergillus niger also precipitated lead oxalate during growth in the presence of lead carbonate, vanadium(V) oxide and ammonium metavanadate, while abiotic tests confirmed the efficacy of oxalic acid in solubilizing vanadinite and precipitating lead as oxalate. Geochemical modelling confirmed the complexity of vanadium speciation, and the significant effect of oxalate. Oxalate–vanadium complexes markedly reduced the vanadinite stability field, with cationic lead(II) and lead oxalate also occurring. In all treatments and geochemical simulations, no other lead vanadate, or vanadium minerals were detected. This research highlights the importance of oxalate in vanadinite bioweathering and suggests a general fungal transformation of lead‐containing apatite group minerals (e.g. vanadinite, pyromorphite, mimetite) by this mechanism. The findings are also relevant to remedial treatments for lead/vanadium contamination, and novel approaches for vanadium recovery.