Engineering Isopropanol Dehydrogenase for Efficient Regeneration of Nicotinamide Cofactors.

Isopropanol dehydrogenase (IPADH) is one of the most attractive options for nicotinamide cofactor regeneration due to its low cost and simple downstream processing. However, poor thermostability and strict cofactor dependency hinder its practical application for bioconversions. In this study, we simultaneously improved the thermostability (433-fold) and catalytic activity (3.3-fold) of IPADH from Brucella suis via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H) by 1.23 × 106-fold. When these variants were employed in three typical bioredox reactions to drive the synthesis of important chiral pharmaceutical building blocks, they outperformed the commonly used cofactor regeneration systems (glucose dehydrogenase [GDH], formate dehydrogenase [FDH], and lactate dehydrogenase [LDH]) with respect to efficiency of cofactor regeneration. Overall, our study provides two promising IPADH variants with complementary cofactor specificities that have great potential for wide applications. IMPORTANCE Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used. Isopropanol dehydrogenase (IPADH) is one of the most attractive biocatalysts for nicotinamide cofactor regeneration. However, poor thermostability and strict cofactor dependency hinder its practical application. In this work, the thermostability and catalytic activity of an IPADH were simultaneously improved via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H). The resultant variants show great potential for regeneration of nicotinamide cofactors, and the engineering strategy might serve as a useful approach for future engineering of other oxidoreductases.

Enzyme cocktail containing NADH regeneration system for efficient bioremediation of oil sludge contamination.

Oil sludge is one kind of toxic and persistent contamination to ecology system from petroleum industry. In order to recycle contaminated sands and reduce environmental impacts at a lower operating cost, enzyme cocktail 21/CbFDH including NADH regeneration system for oily sludge bioremediation was constructed for the first time. The intracellular enzymes of oil-degrading strain Acinetobacter calcoaceticus 21 were prepared and the formate dehydrogenase gene Cbfdh from Candida boidinii was cloned and functionally expressed in E.coli BL21 induced by lactose. The activity and stability of CbFDH was enhanced through self-induction medium optimization using Box-Behnken design. The CbFDH activity was 12.2 times increased and was only decreased 3.9% upon storage at 30 °C for 5 d. The CbFDH increased the degradation rate of oil in high concentration. For the sludge with 10% oil (w/w), the degradation rate achieved 35.6% after 12 h using enzyme 21/CbFDH with the protein ratio of 1:4. The results will provide novel perspectives for creation and operation of petroleum-degrading enzymes involving formate dehydrogenase with higher efficiency and lower cost comparing to current microbial strains or consortium.

Aerobic Dechlorination of Dichloromethane Using Biostimulation Agent BD-C in Continuous and Batch Cultures of Xanthobacter autotrophicus GJ10.

It is important to construct microbiological treatment systems for organic solvent-contaminated water. We developed a continuous culture supplemented with a biostimulation agent named BD-C, which is formulated from canola oil, and Xanthobacter autotrophicus strain GJ10 for an aerobic dichloromethane (DCM)-dechlorinating microorganism. The continuous culture was a chemostat constructed using a 1 L screw-capped bottle containing artificial wastewater medium with 2.0 mM DCM and 1.0% (v/v) BD-C. The expression of genes for DCM metabolism in the dechlorinating aerobe was monitored and analyzed by reverse transcription-quantitative PCR. Strain GJ10 was able to dechlorinate approximately 74% of the DCM in medium supplemented with BD-C during 12 days of incubation. The DCM dechlorination rate was calculated to be 0.11 mM/day. The ΔΔCT method showed that expression of haloalkane dehalogenase increased 5.4 times in the presence of BD-C. Based on batch culture growth tests conducted with mineral salt medium containing three DCM concentrations (0.07, 0.20, 0.43 and 0.65 mM) with BD-C, the apparent maximum specific consumption rate (νmax) and the saturation constant (Ks) determined for DCM degradation in this test were 19.0 nmol/h/CFU and 0.44 mM, respectively. In conclusion, BD-C enhanced the aerobic degradation of DCM by strain GJ10.

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD(+)-dependent formate dehydrogenase.

Succinic acid is a specialty chemical having numerous applications in industrial, pharmaceutical and food uses. One of the major challenges in the succinate fermentation process is eliminating the formation of byproducts. In this study, we describe eliminating byproduct formate and improving succinate productivity by reengineering a high succinate producing E. coli strain SBS550MG-Cms243(pHL413Km). The NAD(+)-dependent formate dehydrogenase gene (fdh1) of Candida boidinii was coexpressed with Lactococcus lactis pyruvate carboxylase (pycA) under the control of Ptrc and PpycA promoters in plasmid pHL413KF1. The newly introduced fdh1 converts 1 mol of formate into 1 mol of NADH and CO2. The reengineered strain SBS550MG-Cms243(pHL413KF1) retains the reducing power of formate through an increase in NADH availability. In anaerobic shake flask fermentations, the parent strain SBS550MG-Cms243(pHL413Km) consumed 99.86 mM glucose and produced 172.38 mM succinate, 16.16 mM formate and 4.42 mM acetate. The FDH bearing strain, SBS550MG-Cms243(pHL413KF1) consumed 98.43 mM glucose and produced 171.80 mM succinate, 1mM formate and 5.78 mM acetate. Furthermore, external formate supplementation to SBS550MG(pHL413KF1) fermentations resulted in about 6% increase in succinate yields as compared to SBS550MG(pHL413Km). In an anaerobic fed-batch bioreactor process, the average glucose consumption rate, succinate productivity, and byproduct formate concentration of SBS550MG(pHL413Km) was 1.40 g/L/h, 1g/L/h, and 17 mM, respectively. Whereas, the average glucose consumption rate, succinate productivity and byproduct formate concentration of SBS550MG(pHL413KF1) was 2 g/L/h, 2 g/L/h, 0-3 mM respectively. A high cell density culture of SBS550MG(pHL413KF1) showed further improvement in succinate productivity with a higher glucose consumption rate. Reduced levels of byproduct formate in succinate fermentation broth would provide an opportunity for reducing the cost associated with downstream processing, purification, and waste disposal.

Improved Candida methylica formate dehydrogenase fermentation through statistical optimization of low-cost culture media.

NAD⁺-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is of use in the regeneration of NAD(P)H coenzymes, and therefore has strong potential for practical application in chemical and medical industries. A low-cost production of recombinant Escherichia coli (E. coli) containing FDH from Candida methylica (cmFDH) was optimized in molasses-based medium by using response surface methodology (RSM) based on central composite design (CCD). The beet molasses as a sole carbon source, (NH4)2HPO4 as a nitrogen and phosphorus source, KH2PO4 as a buffer agent, and Mg2SO4 · 7H2O as a magnesium and sulfur source were used as variables in the medium. The optimum medium composition was found to be 34.694 g L⁻¹ of reducing sugar (equivalent to molasses solution), 8.536 g L⁻¹ of (NH4)2HPO4, 3.073 g L⁻¹ of KH2PO4, and 1.707 g L⁻¹ of Mg2SO4 · 7H2O. Molasses-based culture medium increased the yield of cmFDH about three times compared to LB medium. The currently developed media has the potential to be used in industrial bioprocesses with low-cost production.

Metabolic deficiences revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3).

The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni²âº (Ni²âº-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO4²â» ions could restore hydrogen production to BL21(DE3); however, to only 25-30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO4²â» were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO4²â» and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.

Effects of molybdate and tungstate on expression levels and biochemical characteristics of formate dehydrogenases produced by Desulfovibrio alaskensis NCIMB 13491.

Formate dehydrogenases (FDHs) are enzymes that catalyze the formate oxidation to carbon dioxide and that contain either Mo or W in a mononuclear form in the active site. In the present work, the influence of Mo and W salts on the production of FDH by Desulfovibrio alaskensis NCIMB 13491 was studied. Two different FDHs, one containing W (W-FDH) and a second incorporating either Mo or W (Mo/W-FDH), were purified. Both enzymes were isolated from cells grown in a medium supplemented with 1 µM molybdate, whereas only the W-FDH was purified from cells cultured in medium supplemented with 10 µM tungstate. We demonstrated that the genes encoding the Mo/W-FDH are strongly downregulated by W and slightly upregulated by Mo. Metal effects on the expression level of the genes encoding the W-FDH were less significant. Furthermore, the expression levels of the genes encoding proteins involved in molybdate and tungstate transport are downregulated under the experimental conditions evaluated in this work. The molecular and biochemical properties of these enzymes and the selective incorporation of either Mo or W are discussed.

The changes in pectin metabolism in flax infected with Fusarium.

Fusarium culmorum and Fusarium oxysporum are the most common fungal pathogens of flax (Linum usitatissimum L.), thus leading to the greatest losses in crop yield. A subtractive cDNA library was constructed from flax seedlings exposed for two days to F. oxysporum. This revealed a set of genes that are potentially involved in the flax defense responses. Two of those genes directly participate in cell wall sugar polymer metabolism: UDP-D-glucuronate 4-epimerase (GAE; EC 5.1.3.6) and formate dehydrogenase (FDH; EC 1.2.1.2). GAE delivers the main substrate for pectin biosynthesis, and decreases were detected in its mRNA level after Fusarium infection. FDH participates in the metabolism of formic acid, and the expression level of its gene increased after Fusarium infection. However, metabolite profiling analysis disclosed that the pectin content in the infected plants remained unchanged, but that there were reductions in both the levels of the soluble sugars that serve as pectin precursors, and in the level of formic acid. Since formic acid is the product of pectin demethylesterification, the level of mRNAs coding for pectin methylesterase (EC 3.1.1.11) in the infected flax was measured, revealing a decrease in its expression upon plant infection. Transgenic flax plants overexpressing fungal polygalacturonase (EC 3.2.1.15) and rhamnogalacturonase (EC 3.2.1.-) showed a decrease in the pectin content and an elevated level of formic acid, but the level of expression of the FDH gene remained unchanged. It is suspected that the expression of the formate dehydrogenase gene is directly controlled by the pathogen in the early stage of infection, and additionally by pectin degradation in the later stages.

Transcription of fdh and hyd in Syntrophobacter spp. and Methanospirillum spp. as a diagnostic tool for monitoring anaerobic sludge deprived of molybdenum, tungsten and selenium.

Formate dehydrogenases and hydrogenases contain molybdenum or tungsten and/or selenium. These enzymes are crucial for interspecies formate and hydrogen transfer between propionate degrading Syntrophobacter spp. and methanogenic Methanospirillum spp. Here we used reverse transcription of total RNA followed by quantitative PCR (RT-qPCR) with specific primers to get insight into interspecies formate and hydrogen transfer. Transcriptional regulation of formate dehydrogenases and hydrogenases in Syntrophobacter and Methanospirillum spp. in a propionate-fed up-flow anaerobic sludge bed (UASB) reactor was examined. In both microorganisms formate dehydrogenase and hydrogenase coding genes (fdh and hyd respectively) were transcribed simultaneously. During 249 days in which molybdenum, tungsten and selenium were not supplied to the reactor feed, the microbial activity and transcription of fdh and hyd in Syntrophobacter spp. decreased. Transcription of fdh and hyd in Methanospirillum spp. did not decrease, but transcription of fdh increased when after 249 days molybdenum, tungsten and selenium were supplied to the reactor feed. The developed RT-qPCR is a technique that can give rapid information about active processes in methanogenic granular sludge and may contribute to predict metal limitation and failure in UASB reactors.

Genes for selenium dependent and independent formate dehydrogenase in the gut microbial communities of three lower, wood-feeding termites and a wood-feeding roach.

The bacterial Wood-Ljungdahl pathway for CO(2)-reductive acetogenesis is important for the nutritional mutualism occurring between wood-feeding insects and their hindgut microbiota. A key step in this pathway is the reduction of CO(2) to formate, catalysed by the enzyme formate dehydrogenase (FDH). Putative selenocysteine- (Sec) and cysteine- (Cys) containing paralogues of hydrogenase-linked FDH (FDH(H)) have been identified in the termite gut acetogenic spirochete, Treponema primitia, but knowledge of their relevance in the termite gut environment remains limited. In this study, we designed degenerate PCR primers for FDH(H) genes (fdhF) and assessed fdhF diversity in insect gut bacterial isolates and the gut microbial communities of termites and cockroaches. The insects examined herein represent three wood-feeding termite families, Termopsidae, Kalotermitidae and Rhinotermitidae (phylogenetically 'lower' termite taxa); the wood-feeding roach family Cryptocercidae (the sister taxon to termites); and the omnivorous roach family Blattidae. Sec and Cys FDH(H) variants were identified in every wood-feeding insect but not the omnivorous roach. Of 68 novel alleles obtained from inventories, 66 affiliated phylogenetically with enzymes from T. primitia. These formed two subclades (37 and 29 phylotypes) almost completely comprised of Sec-containing and Cys-containing enzymes respectively. A gut cDNA inventory showed transcription of both variants in the termite Zootermopsis nevadensis (family Termopsidae). The gene patterns suggest that FDH(H) enzymes are important for the CO(2)-reductive metabolism of uncultured acetogenic treponemes and imply that the availability of selenium, a trace element, shaped microbial gene content in the last common ancestor of dictyopteran, wood-feeding insects, and continues to shape it to this day.

Selenium controls transcription of paralogous formate dehydrogenase genes in the termite gut acetogen, Treponema primitia.

The termite gut spirochete, Treponema primitia, is a CO(2)-reductive acetogen that is phylogenetically distinct from other distantly related and more extensively studied acetogens such as Moorella thermoacetica. Research on T. primitia has revealed details about the role of spirochetes in CO(2)-reductive acetogenesis, a process important to the mutualism occurring between termites and their gut microbial communities. Here, a locus of the T. primitia genome containing Wood-Ljungdahl pathway genes for CO(2)-reductive acetogenesis was sequenced. This locus contained methyl-branch genes of the pathway (i.e. for the reduction of CO(2) to the level of methyl-tetrahydrofolate) including paralogous genes for cysteine and selenocysteine (Sec) variants of formate dehydrogenase (FDH) and genes for Sec incorporation. The FDH variants affiliated phylogenetically with hydrogenase-linked FDH enzymes, suggesting that T. primitia FDH enzymes utilize electrons derived directly from molecular H(2). Sub-nanomolar concentrations of selenium decreased transcript levels of the cysteine variant FDH gene. Selenium concentration did not markedly influence the level of mRNA upstream of the Sec-codon in the Sec variant FDH; however, the level of transcript extending downstream of the Sec-codon increased incrementally with increasing selenium concentrations. The features and regulation of these FDH genes are an indication that T. primitia may experience dynamic selenium availability in its H(2)-rich gut environment.

Cloning of a cDNA encoding a NAD-dependent formate dehydrogenase involved in oxalic acid metabolism from the white-rot fungus Ceriporiopsis subvermispora and its gene expression analysis.

The authors have proposed previously that intracellular degradation of oxalic acid via formate to CO(2) occurs in the white-rot fungus Ceriporiopsis subvermispora. The formate degradation is catalyzed by NAD-dependent formate dehydrogenase (CsFDH). In this study, two cDNAs named CsFDH1 and CsFDH2 encoding CsFDH were cloned. Each cDNA consisting of 1077 bp encodes a mature protein composed of 358 amino acid residues. The amino acid sequences of the deduced CsFDH1 and CsFDH2 showed 99% identity to each other. The predicted molecular mass for each was 39.3 kDa, which was similar to that of CsFDH purified from the vegetative mycelia of Ceriporiopsis subvermispora (purified-CsFDH). The recombinant CsFDH1 and CsFDH2 expressed by Escherichia coli showed FDH activity with similar characteristics to the purified CsFDH. However, the amount of CsFDH1 transcript from the vegetative mycelia was 236-691 times greater than that of CsFDH2. Therefore, the results strongly suggest that CsFDH1, as compared with CsFDH2, predominantly contributes to the production of the purified CsFDH.

Dihydroxyacetone detoxification in Saccharomyces cerevisiae involves formaldehyde dissimilation.

To investigate Saccharomyces cerevisiae physiology during growth on the conditionally toxic triose dihydroxyacetone (DHA), protein expression was studied in strains overexpressing either of the two dihydroxyacetone kinase isogenes, DAK1 or DAK2, that grow well utilizing DHA as a carbon and energy source. DHA metabolism was found mostly similar to ethanol utilization, involving a strong component of glucose derepression, but also involved DHA-specific regulatory changes. A specific and strong (10- to 30-fold induction of formaldehyde dehydrogenase, Fdhlp, indicated activation of the formaldehyde dissimilation pathway in DHA medium. The importance of this pathway was further supported by impaired adaptation to DHA growth and DHA survival in a glutathione-dependent formaldehyde dehydrogenase (SFA1) deletion mutant. Glutathione synthase (GSH1) deletion led to decreased DHA survival in agreement with the glutathione cofactor requirement for the SFA1-encoded activity. DHA toxicity did, however, not solely appear related to formaldehyde accumulation, because SFA1 overexpression only enhanced formaldehyde but not DHA tolerance. In further agreement with a low DHA-to-formaldehyde flux, GSH supplements in the low microM range also fully suppressed the DHA sensitivity of a gsh1Delta strain. Under growth reduction on high (100 mM) DHA medium we report increased levels of advanced glycation end-product (AGE) formation on total protein. Under these high-DHA conditions expression of several stress-related proteins, e.g. a heat-shock protein (Hsp104p) and the oxidative stress indicator, alkyl hydroperoxide reductase (Ahp1p) was also found induced. However, hallmark determinants of oxidative stress tolerance (e.g. YAP1, SKN7, HYR1/GPX3 and SOD2) were redundant for DHA tolerance, thus indicating mechanisms of DHA toxicity largely independent of central oxidative stress defence mechanisms. We conclude that mechanisms for DHA growth and detoxification appear complex and that the evolutionary strive to minimize detrimental effects of this intracellular metabolite links to both formaldehyde and glutathione metabolism.

Repression of formate dehydrogenase in Solanum tuberosum increases steady-state levels of formate and accelerates the accumulation of proline in response to osmotic stress.

Formate dehydrogenase (FDH, EC 1.2.1.2.) is a soluble mitochondrial enzyme capable of oxidizing formate into CO2 in the presence of NAD+. It is abundant in non-green tissues and scarce in photosynthetic tissues. Under stress, FDH transcripts (and protein) accumulate in leaves, and leaf mitochondria acquire the ability to use formate as a respiratory substrate. In this paper, we describe the analysis of transgenic potato plants under-expressing FDH, obtained in order to understand the physiological function of FDH. Plants expressing low FDH activities were selected and the study was focused on a line (AS23) showing no detectable FDH activity. AS23 plants were morphologically indistinguishable from control plants, and grew normally under standard conditions. However, mitochondria isolated from AS23 tubers could not use formate as a respiratory substrate. Steady-state levels of formate were higher in AS23 leaves and tubers than in control plants. Tubers of untransformed plants oxidized 14C formate into 14CO2 but AS23 tubers accumulated it. In order to reveal a possible phenotype under stress conditions, control and AS23 plants were submitted to drought and cold. These treatments dramatically induced FDH transcripts in control plants but, whatever the growth conditions, no 1.4 kb FDH transcripts were detected in leaves of AS23 plants. Amongst various biochemical and molecular differences between stressed AS23 and control plants, the most striking was a dramatically faster accumulation of proline in the leaves of drought-stressed plants under-expressing FDH.

Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria.

Most mitochondrial proteins are encoded by the nucleus, translated in the cytosol, and imported. Mitochondrial precursors generally contain their targeting information in a cleavable N-terminal presequence, which is rich in hydroxylated and positively charged residues and can form amphiphilic alpha-helices. We report the in vivo targeting of green fluorescent protein (GFP) by the FDH presequence, as well as several truncated or mutated variants. Some of these mutations modify the amphiphilicity of the predicted alpha-helix. The removal of the first two residues abolishes import and some single amino acid mutations strongly inhibit import. Such strong effects on import had not been observed in similar studies on other plant mitochondrial presequences, suggesting that the FDH presequence is a particularly good model for functional studies.

Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

Inactivation of the selB gene in Methanococcus maripaludis: effect on synthesis of selenoproteins and their sulfur-containing homologs.

The genome of Methanococcus maripaludis harbors genes for at least six selenocysteine-containing proteins and also for homologs that contain a cysteine codon in the position of the UGA selenocysteine codon. To investigate the synthesis and function of both the Se and the S forms, a mutant with an inactivated selB gene was constructed and analyzed. The mutant was unable to synthesize any of the selenoproteins, thus proving that the gene product is the archaeal translation factor (aSelB) specialized for selenocysteine insertion. The wild-type form of M. maripaludis repressed the synthesis of the S forms of selenoproteins, i.e., the selenium-independent alternative system, in selenium-enriched medium, but the mutant did not. We concluded that free selenium is not involved in regulation but rather a successional compound such as selenocysteyl-tRNA or some selenoprotein. Apart from the S forms, several enzymes from the general methanogenic route were affected by selenium supplementation of the wild type or by the selB mutation. Although the growth of M. maripaludis on H(2)/CO(2) is only marginally affected by the selB lesion, the gene is indispensable for growth on formate because M. maripaludis possesses only a selenocysteine-containing formate dehydrogenase.

Overexpression of wild type and SeCys/Cys mutant of human thioredoxin reductase in E. coli: the role of selenocysteine in the catalytic activity.

In contrast to Escherichia coli and yeast thioredoxin reductases, the human placental enzyme contains an additional redox center consisting of a cysteine-selenocysteine pair that precedes the C-terminal glycine residue. This reactive selenocysteine-containing center imbues the enzyme with its unusually wide substrate specificity. For expression of the human gene in E. coli, the sequence corresponding to the SECIS element required for selenocysteine insertion in E. coli formate dehydrogenase H was inserted downstream of the TGA codon in the human thioredoxin reductase gene. Omission of this SECIS element from another construct resulted in termination at UGA. Change of the TGA codon to TGT gave a mutant enzyme form in which selenocysteine was replaced with cysteine. The three gene products were purified using a standard isolation protocol. Binding properties of the three proteins to the affinity resins used for purification and to NADPH were similar. The three proteins occurred as dimers in the native state and exhibited characteristic thiolate-flavin charge transfer spectra upon reduction. With DTNB as substrate, compared to native rat liver thioredoxin reductase, catalytic activities were 16% for the recombinant wild type enzyme, about 5% for the cysteine mutant enzyme, and negligible for the truncated enzyme form.

Production of functional human selenocysteine-containing KDRF/thioredoxin reductase in E. coli.

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.