Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices.
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NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 exhibits the highest stability among the similar type enzymes studied. To obtain further increase in the thermal stability of FDH we used one of general approaches based on hydrophobization of protein alpha-helices. Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studies of nine FDH amino acid sequences from different sources and (ii) with the analysis of the ternary structure of the enzyme from Pseudomonas sp.101. Residues Ser-131 and Ser-160 were replaced by Ala, Val and Leu. Residues Ser-168, Ser-184 and Ser-228 were changed into Ala. Only Ser/Ala mutations in positions 131, 160, 184 and 228 resulted in an increase of the FDH stability. Mutant S168A was 1.7 times less stable than the wild-type FDH. Double mutants S(131,160)A and S(184,228)A and the four-point mutant S(131,160,184,228)A were also prepared and studied. All FDH mutants with a positive stabilization effect had the same kinetic parameters as wild-type enzyme. Depending on the position of the replaced residue, the single point mutation Ser/Ala increased the FDH stability by 5-24%. Combination of mutations shows near additive effect of each mutation to the total FDH stabilization. Four-point mutant S(131,160,184,228)A FDH had 1.5 times higher thermal stability compared to the wild-type enzyme. (+info)
Transcriptional regulation of molybdoenzyme synthesis in Escherichia coli in response to molybdenum: ModE-molybdate, a repressor of the modABCD (molybdate transport) operon is a secondary transcriptional activator for the hyc and nar operons.
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Escherichia coli growing under anaerobic conditions produces several molybdoenzymes, such as formate hydrogenlyase (formate to H2 and CO2; hyc and fdhF genes) and nitrate reductase (narGHJI genes). Synthesis of these molybdoenzymes, even in the presence of the cognate transcriptional activators and effectors, requires molybdate in the medium. Besides the need for molybdopterin cofactor synthesis, molybdate is also required for transcription of the genes encoding these molybdoenzymes. In E. coli, ModE was previously identified as a repressor controlling transcription of the operon encoding molybdate transport components (modABCD). In this work, the ModE protein was also found to be a required component in the activation of hyc-lacZ to an optimum level, but only in the presence of molybdate. Mutant ModE proteins which are molybdate-independent for repression of modA-lacZ also restored hyc-lacZ expression to the wild-type level even in the absence of molybdate. Nitrate-dependent enhancement of transcription of narX-lacZ was completely abolished in a modE mutant. Nitrate-response by narG-lacZ and narK-lacZ was reduced by about 50% in a modE mutant. DNase I footprinting experiments revealed that the ModE protein binds the hyc promoter DNA in the presence of molybdate. ModE-molybdate also protected DNA in the intergenic region between narXL and narK from DNase I hydrolysis. DNA sequences (5' TAYAT 3' and 5' GTTA 3') found in ModE-molybdate-protected modABCD operator DNA were also found in the ModE-molybdate-protected region of hyc promoter DNA (5' GTTA-7 bp-CATAT 3') and narX-narK intergenic region (5' GTTA-7 bp-TACAT 3'). Based on these results, a working model is proposed in which ModE-molybdate serves as a secondary transcriptional activator of both the hyc and narXL operons which are activated primarily by the transcriptional activators, FhlA and NarL, respectively. (+info)
Mapping the cytochrome c553 interacting site using 1H and 15N NMR.
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Cytochrome c553 is the electron transfer partner of formate dehydrogenase and of [Fel-hydrogenase, two metalloenzymes essential in the metabolism of sulfate reducing bacteria. These two enzymes contain a 'ferredoxin-like' domain which presents 30% identity with Desulfovibrio desulfuricans Norway ferredoxin 1. This was chosen as a model for the 'ferredoxin-like' domain involved in the electron transfer reaction with cytochrome c553. ID NMR titration of complex formation gave us the stoichiometry (1:1) and the dissociation constant of the complex (Kd approximately 3x10(-6) M). 2D heteronuclear NMR experiments were performed to analyze the 1H and 15N chemical shift variations that are induced by the protein-protein recognition. This is the first mapping of the interaction site on a c-type cytochrome, using heteronuclear NMR. (+info)
Stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues.
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The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site-directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the Tm values revealed that the stability at temperatures above 50 degrees C is optimal for the native and the recombinant wt enzyme (Tm 57 degrees C), whereas the Tm values of the mutant enzymes vary in the range 44-52 degrees C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to Ltert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme. (+info)
Dissection of the maturation reactions of the [NiFe] hydrogenase 3 from Escherichia coli taking place after nickel incorporation.
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The steps in the maturation of the precursor of the large subunit (pre-HycE) of hydrogenase 3 from Escherichia coli taking place after incorporation of both iron and nickel were investigated. Pre-HycE could be matured and processed in the absence of the small subunit but association with the cytoplasmic membrane required heterodimer formation between the two subunits. Pre-HycE formed a complex with the chaperone-like protein HypC in the absence of the small subunit and, in this complex, also incorporated nickel. For the C-terminal processing, HypC had to leave the complex since only a HypC-free, nickel-containing form of pre-HycE was a substrate for the maturation endopeptidase. (+info)
Production of functional human selenocysteine-containing KDRF/thioredoxin reductase in E. coli.
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In a previous study, we reported the isolation of a cDNA encoding KDRF (KM-102-derived reductase like factor) from the human bone marrow-derived stromal cell line KM-102. Analysis of the sequence of this cDNA revealed it to be the previously reported human thioredoxin reductase cDNA. Human thioredoxin reductase, which was recently isolated from human lung adenocarcinoma NCI-H441 cells as a selenocysteine-containing selenoprotein, and its substrate thioredoxin are thought to be essential for protecting cells from the damage caused by reactive oxygen species. To obtain the selenocysteine-containing recombinant KDRF/thioredoxin reductase, we introduced a secondary structure, which is identical to the selenocysteine insertion signal of Escherichia coli formate dehydrogenase H mRNA, downstream of the TGA in the KDRF/thioredoxin reductase cDNA and expressed it in E. coli. As a result, a significant amount of selenocysteine was incorporated into the C-terminus of the KDRF/thioredoxin reductase protein. The selenocysteine-containing KDRF/thioredoxin reductase showed reducing activities toward human and E. coli thioredoxin, whereas non-selenocysteine-containing KDRF/thioredoxin reductase showed no enzyme activity. Our results suggest that this strategy will be applicable to the production of other mammalian selenocysteine-containing selenoproteins in E. coli. (+info)
Effects of the Calvin cycle on nicotinamide adenine dinucleotide concentrations and redox balances of Xanthobacter flavus.
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The levels of reduced and oxidized nicotinamide adenine dinucleotides were determined in Xanthobacter flavus during a transition from heterotrophic to autotrophic growth. Excess reducing equivalents are rapidly dissipated following induction of the Calvin cycle, indicating that the Calvin cycle serves as a sink for excess reducing equivalents. The physiological data support the conclusion previously derived from molecular studies in that expression of the Calvin cycle genes is controlled by the intracellular concentration of NADPH. (+info)
Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii.
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We isolated the promoter regions of five methanol-inducible genes (P(AOD1), alcohol oxidase; P(DAS1), dihydroxyacetone synthase; P(FDH1), formate dehydrogenase; P(PMP20), Pmp20; and P(PMP47), Pmp47) from the Candida boidinii genome, and evaluated their strength and studied their regulation using the acid phosphatase gene of Saccharomyces cerevisiae (ScPHO5) as the reporter. Of the five promoters, P(DAS1) was the strongest methanol-inducible promoter whose strength was approximately 1.5 times higher than that of the commonly used P(AOD1) in methanol-induced cells. Although the expression of P(AOD1) and P(DAS1) was completely repressed by the presence of glucose, formate-induced expression of P(FDH1) was not repressed by glucose. Expression under P(PMP47), another methanol-inducible promoter, was highly induced by oleate. The induction kinetics of P(PMP47) and P(DAS1) revealed that methanol induces the expression of peroxisome membrane protein Pmp47, earlier than the expression of matrix enzyme dihydroxyacetone synthase (Das1p), and that this information is contained in the promoter region of the respective gene. This is the first report which evaluates several methanol-inducible promoters in parallel in the methylotrophic yeast. (+info)