Factor 420-dependent tyridine nucleotide-linked hydrogenase system of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.     

Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.     

Purification and properties of F420- and NADP(+)-dependent alcohol dehydrogenases of Methanogenium liminatans and Methanobacterium palustre, specific for secondary alcohols

The F420-dependent alcohol dehydrogenase (ADH) of Methanogenium liminatans and the NADP(+)-dependent ADH of Methanobacterium palustre were purified to homogeneity. The native F420-dependent ADH of Mg. liminatans had a molecular mass of 150 kDa and consisted of four (presumably identical) subunits with a mass of 39 kDa. The temperature optimum was 42 degrees C, the optimum pH 6.0 and NaCl or KCl were inhibitory. The NADP(+)-dependent ADH of Mb. palustre had a molecular mass of 175 kDa and consisted also of four (presumably identical) subunits with a mass of 44 kDa. The temperature optimum was 60 degrees C, the optimum pH 8.0 and optimal activity was observed in the presence of 500 mM NaCl or KCl. The ADHs of both organisms catalysed the oxidation of various secondary and cyclic alcohols to the corresponding ketones and the reverse reaction. No primary alcohols were apparently oxidized. The NADP(+)-dependent ADH of Mb. palustre contained 4-8 mol atoms zinc/mol enzyme and was inhibited by low concentrations of iodoacetate and 4-hydroxymercuribenzoate, whereas the F420-dependent ADH of Mg. liminatans presumably contained no zinc ions and was inhibited by 1,10-phenanthroline or high concentrations (e.g. 100 microM) of 4-hydroxymercuribenzoate. Polyclonal antibodies against the NADP(+)-dependent ADH of Mb. palustre precipitated only the homologous ADH. A precipitation of the NADP(+)-dependent ADH of Methanocorpusculum parvum required a 10-fold higher antibody concentration, showing at least a distant relationship of both ADHs. Antibodies against the NADP(+)-dependent ADH of Mcp. parvum, however, formed precipitates with the homologous ADH of Mcp. parvum and with the NADP(+)-dependent ADH of Mb. palustre. They also formed precipitates with the ADH of Thermoanaerobium brockii, which is not related to methane bacteria. Antibodies against the F420-dependent ADH of Mg. liminatans reacted only with the homologous enzyme and did not form precipitates with NADP(+)-dependent ADHs. No immunological relation of the NADP(+)- or F420-dependent ADHs of methanogens with ADH of yeast or horse liver was found. In accordance with the immunological data, the N-terminal amino acid sequences of the NADP(+)-dependent ADHs of Mb. palustre and Mcp. parvum had a high degree of similarity, whereas the N-terminal amino acid sequence of the ADH of Mg. liminatans revealed no similarity with the two NADP(+)-dependent enzymes.     

Modeling and phylogenetic analysis of cytosolic ascorbate peroxidase (OsAPX1) from rice reveal signature motifs that may play a role in stress tolerance

Ascorbate peroxidase (APX) is a crucial, haeme-containing enzyme of the ascorbate glutathione cycle that detoxifies reactive oxygen species in plants by catalyzing the conversion of hydrogen peroxide to water using ascorbate as a specific electron donor. Different APX isoforms are present in discrete subcellular compartments in rice and their expression is stress regulated. We revealed the homology model of OsAPX1 protein using the crystal structure of soybean GmAPX1 (PDB ID: 2XIF) as template by Modeller 9.12. The resultant OsAPX1 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that indicated the model structure is reliable with 83 % amino acid sequence identity with template, RMSD (1.4 Å), Verify3D (86.06 %), Zscores (-8.44) and Ramachandran plot analysis showed that conformations for 94.6% of amino acid residues are within the most favoured regions. Investigation revealed two conserved signatures for haeme ligand binding and peroxidase activity in the alpha helical region that may play a significant role during stress.     

Structure of the F420H2:quinone oxidoreductase of Archaeoglobus fulgidus identification and overproduction of the F420H2-oxidizing subunit.

The F420H2:quinone oxidoreductase from the sulfate-reducing archaeon Archaeoglobus fulgidus is encoded by the fqo gene cluster which comprises 11 genes (fqo J, K, M, L, N, A, BC, D, H, I, F). The last gene of the cluster, fqoF, was overexpressed in Escherichia coli. The purified subunit was able to oxidize reduced cofactor F420 using the electron-acceptor system methyl viologen plus metronidazole. The specific activity at 78 degrees C was 64 micromol F420H2 oxidized. min-1.(mg protein)-1. The purified polypeptide contained 10.6 mol non-heme iron, 7.2 mol acid-labile sulfur and 0.7 mol FAD per mol protein. With the exception of fqoF, the deduced amino-acid sequences of all other genes show homologies to distinct subunits of NADH-quinone oxidoreductases from prokaryotes and eukaryotes. Thus, it is concluded that the F420H2-dependent and the NADH-dependent enzyme are functional equivalents. Both proteins are the initial enzymes of membrane-bound electron-transport systems and are involved in energy conservation. In parallel with bacterial complex I, the F420H2:quinone oxidoreductase may be composed of three subcomplexes. FqoF functions as the input device adjusted to the oxidation of reduced cofactor F420H2, thereby replacing subunits of the input module of complex I that are not present in A. fulgidus. The subunits FqoB, FqoCD and FqoI may form the membrane-associated module and transfer electrons to the membrane-integral module. It is most likely that the last subcomplex is composed of FqoA, FqoH, FqoJ, FqoK, FqoL, FqoM and FqoN. All subunits are highly hydrophobic and are probably involved in the reduction of a special menaquinone with a fully reduced isoprenoid side chain present in the cytoplasmic membrane of A. fulgidus.     

Structures of F420H2:NADP+ oxidoreductase with and without its substrates bound.

Cofactor F420 is a 5'-deazaflavin derivative first discovered in methanogenic archaea but later found also to be present in some bacteria. As a coenzyme, it is involved in hydride transfer reactions and as a prosthetic group in the DNA photolyase reaction. We report here for the first time on the crystal structure of an F420-dependent oxidoreductase bound with F420. The structure of F420H2:NADP+ oxidoreductase resolved to 1.65 A contains two domains: an N-terminal domain characteristic of a dinucleotide-binding Rossmann fold and a smaller C-terminal domain. The nicotinamide and the deazaflavin part of the two coenzymes are bound in the cleft between the domains such that the Si-faces of both face each other at a distance of 3.1 A, which is optimal for hydride transfer. Comparison of the structures bound with and without substrates reveals that of the two substrates NADP has to bind first, the binding being associated with an induced fit.     

Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.     

Phylogenetic analysis of methanogens in sheep rumen ecosystem and detection of Methanomicrobium mobile By fluorescence in situ hybridization

The population of methanogens in the sheep rumen microbial ecosystem was studied by using 16S rDNA cloning analysis, epifluorescence microscopy (which detects autofluorescence of a specific cofactor F420 in methanogens) and the 16S rRNA-targeted in situ hybridization technique. The 16S rDNA clone libraries were constructed by PCR amplification with an Archaea-specific primer set and partial sequencing of the clonal 16S rDNAs was done. Phylogenetic analysis indicated that the clones were affiliated with Methanomicrobium ruminantium and mobile, Methanobrevibacter smithii. Epifluorescence microscopy (F420 autofluorescence) and in situ hybridization by using a newly designed M. mobile-specific 16S rRNA-targeted oligonucleotide probe found that methanogens accounted for approximately 3.6% of total ruminal microorganisms and approximately 54% of the total methanogens were M. mobile.     

Function of coenzyme F420 in aerobic catabolism of 2,4, 6-trinitrophenol and 2,4-dinitrophenol by Nocardioides simplex FJ2-1A

2,4,6-Trinitrophenol (picric acid) and 2,4-dinitrophenol were readily biodegraded by the strain Nocardioides simplex FJ2-1A. Aerobic bacterial degradation of these pi-electron-deficient aromatic compounds is initiated by hydrogenation at the aromatic ring. A two-component enzyme system was identified which catalyzes hydride transfer to picric acid and 2,4-dinitrophenol. Enzymatic activity was dependent on NADPH and coenzyme F420. The latter could be replaced by an authentic preparation of coenzyme F420 from Methanobacterium thermoautotrophicum. One of the protein components functions as a NADPH-dependent F420 reductase. A second component is a hydride transferase which transfers hydride from reduced coenzyme F420 to the aromatic system of the nitrophenols. The N-terminal sequence of the F420 reductase showed high homology with an F420-dependent NADP reductase found in archaea. In contrast, no N-terminal similarity to any known protein was found for the hydride-transferring enzyme.     

Factor 420-dependent pyridine nucleotide-linked formate metabolism of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a formate dehydrogenase which is linked to factor 420 (F420) as the first low-molecular-weight or anionic electron transfer coenzyme. Reduced F420 obtained from the formate dehydrogenase can be further linked to the formation of hydrogen via the previously described F420-dependent hydrogenase reaction, thus constituting an apparently simple formate hydrogenlyase system, or to the reduction of nicotinamide adenine dinucleotide phosphate via F420:nicotinamide adenine dinucleotide phosphate oxidoreductase. The results indicate that hydrogen and formate, the only known energy sources for M. ruminantium and many other methanogenic bacteria, should be essentially equivalent as sources of electrons in the metabolism of this organism.     

Identification of potential Leptospira phosphoheptose isomerase inhibitors through virtual high-throughput screening

The hfe-threatening infections caused by Leptospira serovars demand the need for designing anti-leptospirosis drugs.The present study encompasses exploring inhibitors against phosphoheptose isomerase(GmhA)of Leptospira,which is vital for lipopolysaccharide(LPS)biosynthesis and is identified as a common drug target through the subtractive genomic approach.GmhA model was built in Modeller 9v7.Structural refinement and energy minimization of the predicted model was carried out using Maestro 9.0.The refined model reliability was assessed through Procheck,ProSA,ProQ and Profile 3D.The substrate-based virtual high-throughput screening(VHTS)in Ligand.Info Meta-Database tool generated an in-house library of 354 substrate structural analogs.Furthermore,structure-based VHTS from the in-house library with different conformations of each ligand provided 14 novel competitive inhibitors.The model together with insight gained from the VHTS would be a promising starting point for developing anti-leptospirosis competitive inhibitors targeting LPS biosynthesis pathway.     

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B.

The capacities of Procion Red HE-3B and Cibacron Blue F3G-A immobilized to Sepharose CL-4B and Matrex 201R for NAD+-, NADP+- and NAD(P)+-dependent dehydrogenases were measured. Procion Red HE-3B columns retarded NADP+-dependent dehydrogenases more effectively than NAD+-dependent dehydrogenases, whilst immobilized Cibacron Blue F3G-A retarded NAD+-dependent dehydrogenases more effectively than NADP+-dependent dehydrogenases. The capacity of procion Red HE-3B-Sepharose CL-4B for five dehydrogenases was highest in the region of 70nmol of immobilized ligand/ml of settled gel. The effects of using poly(ethyleneimine) as a spacer for both porous and pellicular supports were also examined. Four NADP+-dependent dehydrogenases were purified from yeast extract by using Procion Red HE-3B-Sepharose CL-4B. Two NAD+-dependent dehydrogenases were purified from the same source using Cibacron Blue F3G-A-Sepharose CL-4B. These results are discussed in relation to the use of immobilized Procion Red HE-3B to purify dehydrogenases. This immobilized dye's chromatograhic behaviour is compared with that of immobilized nucleotides. The most important feature of immobilized tirazine dyes seems to be their high operational capacities when compared with group-specific nucleotide adsorbents.     

Homology modeling and in silico screening of inhibitors for the substrate binding domain of human Siah2: implications for hypoxia-induced cancers

The three-dimensional (3D) structure of the substrate binding domain (SBD) of human ubiquitin ligase Siah2 (seven in absentia homolog) was constructed based on the homology modeling approach using the Modeller 9v7 program. The molecular dynamics method was utilized to refine the model and it was further assessed by ProSA, three-dimensional structural superposition (3d-SS) and PROCHEK in order to analyze the quality and reliability of the generated model. Furthermore, we predicted the binding pocket of Siah2 and also validated it by both blind and normal docking using a known functional inhibitor, menadione. Using structure-based high-throughput virtual screening, we identified five lead drug-like molecules against the modeled SBD of Siah2 and analyzed its pharmacokinetic properties to identify the potential inhibitors for Siah2. The docking results for menadione and the lead molecules at the ligand binding site of SBD of Siah2 revealed that the residue Ser39 (corresponding to Ser167 in the full-length protein) is consistently involved in strong hydrogen bonding, and plays an important role in phosphorylation and the enhanced activity of Siah2.     

insilico Characterization and Homology Modeling of Arabitol Dehydrogenase (ArDH) from Candida albican

Background: Arabitol dehydrogenase (ArDH) is involved in the production of different sugar alcohols like arabitol, sorbitol, mannitol, erythritol and xylitol by using five carbon sugars as substrate. Arabinose, d-ribose, d-ribulose, xylose and d-xylulose are known substrate of this enzyme. ArDH is mainly produced by osmophilic fungi for the conversion of ribulose to arabitol under stress conditions. Recently this enzyme has been used by various industries for the production of pharmaceutically important sugar alcohols form cheap source than glucose. But the information at structure level as well as its binding energy analysis with different substrates was missing. Results: The present study was focused on sequence analysis, insilico characterization and substrate binding analysis of ArDH from a fungus specie candida albican. Sequence analysis and physicochemical properties showed that this protein is highly stable, negatively charged and having more hydrophilic regions, these properties made this enzyme to bind with number of five carbon sugars as substrate. The predicted 3D model will helpful for further structure based studies. Docking analysis provided free energies of binding of each substrate from a best pose as arabinose -9.8224calK/mol, dribose -11.3701Kcal/mol, d-ribulose -8.9230Kcal/mol, xylose -9.7007Kcal/mol and d-xylulose 9.7802Kcal/mol. Conclusion: Our study provided insight information of structure and interactions of ArDH with its substrate. These results obtained from this study clearly indicate that d-ribose is best substrate for ArDH for the production of sugar alcohols. This information will be helpful for better usage of this enzyme for hyper-production of sugar alcohols by different industries.     

Sugar utilization in the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324: starch degradation to acetate and CO2 via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming)

The hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324, rather than the type strain VC16, was found to grow on starch and sulfate as energy and carbon source. Fermentation products and enzyme activities were determined in starch-grown cells and compared to those of cells grown on lactate and sulfate. During exponential growth on starch, 1 mol of glucose-equivalent was incompletely oxidized with sulfate to approximately 2 mol acetate, 2 mol CO2 and 1 mol H2S. Starch-grown cells did not contain measurable amounts of the deazaflavin factor F420 (<0.03 nmol/mg protein) and thus did not show the F420-specific green-blue fluorescence. In contrast, lactate (1 mol) was completely oxidized with sulfate to 3 mol CO2 by strain 7324, and lactate-grown cells contained high amounts of F420 (0.6 nmol/mg protein). In extracts of starch-grown cells, the following enzymes of a modified Embden-Meyerhof pathway were detected: ADP-dependent hexokinase (ADP-HK), phosphoglucose isomerase, ADP-dependent 6-phosphofructokinase (ADP-PFK), fructose-1,6-phosphate aldolase, glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAP:FdOR), phosphoglycerate mutase, enolase, and pyruvate kinase (PK). Specific activities of ADP-HK, ADP-PFK, GAP:FdOR, and PK were significantly higher in starch-grown cells than in lactate-grown cells, indicating induction of these enzymes during starch catabolism. Pyruvate conversion to acetate involved pyruvate:ferredoxin oxidoreductase and ADP-forming acetyl-CoA synthetase. The findings indicate that the archaeal sulfate reducer A. fulgidus strain 7324 converts starch to acetate via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming). This is the first report of growth of a sulfate reducer on starch, i.e. on a polymeric sugar.     

A Homology Based Model and Virtual Screening of Inhibitors for Human Geranylgeranyl Transferase 1 (GGTase1)

Protein prenylation is a post translational modification that is indispensable for Ras–Rho mediated tumorigenesis. In mammals, three enzymes namely protein farnesyltransferase (FTase), geranylgeranyl transferase1 (GGTase1), and geranylgeranyl transferase2 (GGTase2) were found to be involved in this process. Usually proteins of Ras family will be farnesylated by FTase, Rho family will be geranylgeranylated by GGTase1. GGTase2 is exclusive for geranylgeranylating Rab protein family. FTase inhibitors such as FTI- 277 are potent anti-cancer agents in vitro. In vivo, mutated Ras proteins can either improve their affinity for FTase active site or undergo geranylgeranylation which confers resistance and no activity of FTase inhibitors. This led to the development of GGTase1 inhibitors. A well-defined 3-D structure of human GGTase1 protein is lacking which impairs its in silico and rational designing of inhibitors. A 3-D structure of human GGTase1 was constructed based on primary sequence available and homology modeling to which pubchem molecules library was virtually screened through AutoDock Vina. Our studies show that natural compounds Camptothecin (-8.2 Kcal/mol), Curcumin (-7.3 Kcal/mol) have higher binding affinities to GGTase-1 than that of established peptidomimetic GGTase-1 inhibitors such as GGTI-297 (-7.5 Kcal/mol), GGTI-298 (-7.5 Kcal/mol), CHEMBL525185 (-7.2 Kcal/mol).     

Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. Thermal denaturation and activation.

Pyrococcus furiosus is a marine hyperthermophile that grows optimally at 100 degrees C. Glutamate dehydrogenase (GDH) from P. furiosus is a hexamer of identical subunits and has an M(r) = 270,000 +/- 5500 at 25 degrees C. Electron micrographs showed that the subunit arrangement is similar to that of GDH from bovine liver (i.e. 3/2 symmetry in the form of a triangular antiprism). However, GDH from P. furiosus is inactive at temperatures below 40 degrees C and undergoes heat activation above 40 degrees C. Both NAD+ and NADP+ are utilized as cofactors. Apparently the inactive enzyme also binds cofactors, since the enzyme maintains the ability to bind to an affinity column (Cibacron blue F3GA) and is specifically eluted with NADP+. Conformational changes that accompany activation and thermal denaturation were detected by precision differential scanning microcalorimetry. Thermal denaturation starts at 110 degrees C and is completed at 118 degrees C. delta(cal) = 414 Kcal [mol GDH]-1. Tm = 113 degrees C. This increase in heat capacity indicates an extensive irreversible unfolding of the secondary structure as evidenced also by a sharp increase in absorbance at 280 nm and inactivation of the enzyme. The process of heat activation of GDH from 40 to 80 degrees C is accompanied by a much smaller increase in absorbance at 280 nm and a reversible increase in heat capacity with delta(cal) = 187 Kcal [mol GDH]-1 and Tm = 57 degrees C. This absorbance change as well as the moderate increase in heat capacity suggest that thermal activation leads to some exposure of hydrophobic groups to solvent water as the GDH structure is opened slightly. The increase in absorbance at 280 nm during activation is only 12% of that for denaturation. Overall, GDH appears to be well adapted to correspond with the growth response of P. furiosus to temperature.     

Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc

Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+).     

Si-face stereospecificity at C5 of coenzyme F420 for F420H2 oxidase from methanogenic Archaea as determined by mass spectrometry

Coenzyme F420 is a 5-deazaflavin. Upon reduction, 1,5 dihydro-coenzyme F420 is formed with a prochiral centre at C5. All the coenzyme F420-dependent enzymes investigated to date have been shown to be Si-face stereospecific with respect to C5 of the deazaflavin, despite most F420-dependent enzymes being unrelated phylogenetically. In this study, we report that the recently discovered F420H2 oxidase from methanogenic Archaea is also Si-face stereospecific. The enzyme was found to catalyse the oxidation of (5S)-[5-2H1]F420H2 with O2 to [5-1H]F420 rather than to [5-2H]F420 as determined by MALDI-TOF MS. (5S)-[5-2H1]F420H2 was generated by stereospecific enzymatic reduction of F420 with (14a-2H2)-[14a-2H2] methylenetetrahydromethanopterin.     

A F420-dependent NADP reductase in the extremely thermophilic sulfate-reducing Archaeoglobus fulgidus

Archaeoglobus fulgidus, a sulfate-reducing Archaeon with a growth temperature optimum of 83°C, uses the 5-deazaflavin coenzyme F₄₂₀ rather than pyridine nucleotides in catabolic redox processes. The organism does, however, require reduced pyridine nuclcotides for biosynthetic purposes. We describe here that the Archaeon contains a coenzyme F₄₂₀-dependent NADP reductase which links anabolism to catabolism. The highly thermostable enzyme was purfied 3600-fold by affinity chromatography to apparent homogeneity in a 60% yield. The native enzyme with an apparent molecular mass of 55 kDa was composed of only one type of subunit of apparent molecular mass of 28 kDa. Spectroscopic analysis of the enzyme did not reveal the presence of any chromophoric prosthetic group. The purified enzyme catalyzed the reversible reduction of NADP (apparent K _M 40 M) with reduced F₄₂₀ (apparent K _M 20M) with a specific activity of 660 U/mg (apparent V _max) at pH 8.0 (pH optimum) and 80°C (temperature optimum). It was specific for both coenzyme F₄₂₀ and NADP. Sterochemical investigations showed that the F₄₂₀-dependent NADP reductase was Si face specific with respect to C5 of F₄₂₀ and Si face specific with respect to C4 of NADP.Abbreviations F₄₂₀ coenzyme F₄₂₀ - F₄₂₀H₂ 1,5-dihydrocoenzyme F₄₂₀ - H₄MPT tetrahydromethanopterin - CH=H₄MPT N⁵, N¹⁰-methylenetetrahydromethanopterin - MFR methanofuran - HPLC high performance liquid chromatography - methylene-H₄MPT dehydrogenase N⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase - 1 U = 1 mol/min