A trimeric supercomplex of the oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha H16

The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit.     

Metabolite profiles of polyhydroxyalkanoate-producing Ralstonia eutropha H16

This study describes metabolite profiles of Ralstonia eutropha H16 focusing on biosynthesis of polyhydroxyalkanoates (PHAs), bacterial polyesters attracted as biodegradable bio-based plastics. As CoA-thioesters are important intermediates in PHA biosynthesis, four kinds of acyl-CoAs with medium chain length were prepared and used to establish analytical conditions for capillary electrophoresis-electron spray ionization-tandem mass spectrometry (CE–ESI-MS/MS). Metabolites were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose and PHA production phase on octanoate, and subjected to stable isotope dilution-based comparative quantification by multiple reaction monitoring using CE–ESI-MS/MS and ¹³C-labeled metabolites prepared by extraction from R. eutropha mutant grown on U-¹³C₆-glucose. This procedure allowed to quantify relative changes of 94 ionic metabolites including CoA-thioesters. Hexose-phosphates except for glucose 1-phosphate were decreased in the PHA production phase than in the growth phase, suggesting reduced flux of sugar degradation after the cell growth. Several intermediates in TCA cycle and gluconeogenesis were increased in the PHA production phase on octanoate. Interestingly, ribulose 1,5-bisphosphate were detected in all the samples examined, raising possibilities of CO₂ fixation by Calvin–Benson–Bassham cycle in this bacterium even under heterotrophic growth conditions. Turnover of acyl moieties through β-oxidation was suggested to be active on fructose, as CoA-thioesters of C₆ and C₈ were detected in the fructose-grown cells. In addition, major metabolic pools in R. eutropha cells were estimated from the signal intensities. The results of the present study provided new insights into global metabolisms in PHA-producing R. eutropha.     

The maturation factors HoxR and HoxT contribute to oxygen tolerance of membrane-bound [NiFe] hydrogenase in Ralstonia eutropha H16

The membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O(2) and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O(2) levels. MBH-driven growth on H(2) is inhibited or retarded at high O(2) partial pressure (pO(2)) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO(2). Lack of hoxR and hoxT can phenotypically be restored by providing O(2)-limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions.     

Characterization of propionate CoA-transferase from Ralstonia eutropha H16

In this study, a propionate CoA-transferase (H16_A2718; EC 2.8.3.1) from Ralstonia eutropha H16 (Pct _Re ) was characterized in detail. Glu342 was identified as catalytically active amino acid residue via site-directed mutagenesis. Activity of Pct _Re was irreversibly lost after the treatment with NaBH₄ in the presence of acetyl-CoA as it is shown for all CoA-transferases from class I, thereby confirming the formation of the covalent enzyme-CoA intermediate by Pct _Re . In addition to already known CoA acceptors for Pct _Re such as 3-hydroxypropionate, 3-hydroxybutyrate, acrylate, succinate, lactate, butyrate, crotonate and 4-hydroxybutyrate, it was found that glycolate, chloropropionate, acetoacetate, valerate, trans-2,3-pentenoate, isovalerate, hexanoate, octanoate and trans-2,3-octenoate formed also corresponding CoA-thioesters after incubation with acetyl-CoA and Pct _Re . Isobutyrate was found to be preferentially used as CoA acceptor amongst other carboxylates tested in this study. In contrast, no products were detected with acetyl-CoA and formiate, bromopropionate, glycine, pyruvate, 2-hydroxybutyrate, malonate, fumarate, itaconate, β-alanine, γ-aminobutyrate, levulate, glutarate or adipate as potential CoA acceptor. Amongst CoA donors, butyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, isobutyryl-CoA, succinyl-CoA and valeryl-CoA apart from already known propionyl-CoA and acetyl-CoA could also donate CoA to acetate. The highest rate of the reaction was observed with 3-hydroxybutyryl-CoA (2.5 μmol mg^?1 min^?1). K _m values for propionyl-CoA, acetyl-CoA, acetate and 3-hydroxybutyrate were 0.3, 0.6, 4.5 and 4.3 mM, respectively. The rather broad substrate range might be a good starting point for enzyme engineering approaches and for the application of Pct _Re in biotechnological polyester production.     

Novel,Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H₂. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H₂ uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H₂. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H₂ per min and mg of protein. However, an unexpectedly high Michaelis constant (K_m) for H₂ of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low K_m of group 5 hydrogenases and makes atmospheric H₂ uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H₂ oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O₂.     

Genomic view of energy metabolism in Ralstonia eutropha H16

Ralstonia eutropha is a strictly respiratory facultative lithoautotrophic beta-proteobacterium. In the absence of organic substrates, H2 and CO2 are used as sole sources of energy and carbon. In the absence of oxygen, the organism can respire by denitrification. The recent determination of the complete genome sequence of strain H16 provides the opportunity to reconcile the results of previous physiological and biochemical studies in light of the coding capacity. These analyses revealed genes for several isoenzymes, permit assignment of well-known physiological functions to previously unidentified genes, and suggest the presence of unknown components of energy metabolism. The respiratory chain is fueled by two NADH dehydrogenases, two uptake hydrogenases and at least three formate dehydrogenases. The presence of genes for five quinol oxidases and three cytochrome oxidases indicates that the aerobic respiration chain adapts to varying concentrations of dioxygen. Several additional components may act in balancing or dissipation of redox energy. Paralogous sets of nitrate reductase and nitric oxide reductase genes result in enzymatic redundancy for denitrification.     

The role of the active site-coordinating cysteine residues in the maturation of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16

The H₂-splitting active site of [NiFe] hydrogenases is tightly bound to the protein matrix via four conserved cysteine residues. In this study, the nickel-binding cysteine residues of HoxC, the large subunit of the H₂-sensing regulatory hydrogenase (RH) from Ralstonia eutropha, were replaced by serine. All four mutant proteins, C60S, C63S, C479S, and C482S, were inactive both in H₂ sensing and H₂ oxidation and did not adopt the native oligomeric structure of the RH. Nickel was bound only to the C482S derivative. The assembly of the [NiFe] active site is a complex process that requires the function of at least six accessory proteins. Among these proteins, HypC has been shown to act as a chaperone for the large subunit during the maturation process. Immunoblot analysis revealed the presence of a strong RH-dependent HypC-specific complex in extracts containing the C60S, C63S, and C482S derivatives, pointing to a block in maturation for these mutant proteins. The lack of this complex in the extract containing C479S indicates that this specific cysteine residue might be crucial for the interaction between HoxC and HypC.This work is dedicated to Prof. H.G. Schlegel on the occasion of his 80th birthday.     

Chaperones specific for the membrane-bound [NiFe]-hydrogenase interact with the Tat signal peptide of the small subunit precursor in Ralstonia eutropha H16

Periplasmic membrane-bound [NiFe]-hydrogenases undergo a complex maturation pathway, including cofactor incorporation, subunit assembly, and finally twin-arginine-dependent membrane translocation (Tat). In this study, the role of the two accessory proteins HoxO and HoxQ in the maturation of the membrane-bound [NiFe]-hydrogenase (MBH) of Ralstonia eutropha H16 was investigated. MBH activity was absent in soluble as well as membrane fractions of cells with deletions in the respective genes. The absence of HoxO and HoxQ led to degradation of the small subunit precursor (preHoxK) of the MBH. The two accessory proteins directly interacted with preHoxK prior to assembly of active MBH dimer in the cytoplasm. MBH mutants with modified Tat signal peptides were disrupted in preHoxK/HoxO/HoxQ complex formation. Isolated HoxO and HoxQ proteins formed a complex in vitro with the chemically synthesized HoxK Tat signal peptide. Two functions of the two chaperones are discussed: (i) protection of the Fe-S cluster containing HoxK subunit under oxygenic conditions, and (ii) avoidance of HoxK export prior to dimerization with the large MBH subunit HoxG.     

Systematic evaluation of the dihydrogen-oxidising and NAD+-reducing soluble [NiFe]-hydrogenase from Ralstonia eutropha H16 as a cofactor regeneration catalyst

Abstract

The oxygen-tolerant NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 has been described as a promising catalyst for cofactor regeneration in biocatalysed reductions. In this study, the actual potential of this enzyme for application in technical synthesis was evaluated. An overproduced, purified version of the enzyme was coupled to the carbonyl reductase from Candida parapsilosis (CPCR), where it allowed an almost quantitative conversion of the model substrate; total turnover numbers (TTN: n_product/n_enzyme) of up to 143,666 were achieved. This was distinctly superior to the commonly used NADH regenerating enzyme formate dehydrogenase (FDH) from Candida boidinii. In a systematic quantitative approach, maximum activity for NAD⁺ reduction was observed at 35 °C and pH 8, which corresponds to that of native SH. The half-life of the enzyme under these conditions was 5.3 hours. In the presence of sodium salts, distinct inhibitory effects were observed while ammonium and potassium ions increased the enzyme stability. Overall, a high but not unusual sensitivity of SH for changes in temperature, pH and mechanical stress in a reactor was found. Technical application in chemical synthesis can therefore be considered a feasible goal.     

Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.     

The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).     

Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates

Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin (glpF) and glycerol kinase (glpK) from Escherichia coli. Although introduction of glpFK _Ec into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK _Ec within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK _Ec alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK _Ec or glpK _Ec produced large amounts of poly[(R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK _Ec was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol.     

Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H₂ and CO₂ under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO₂ as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO₂ and H₂ as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.     

Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16

The β-proteobacterium Ralstonia eutropha H16 utilizes fructose and gluconate as carbon sources for heterotrophic growth exclusively via the Entner–Doudoroff pathway with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the responsible gene eda, we constructed a KDPG aldolase-negative strain, which is disabled to supply pyruvate for energy metabolism from fructose or gluconate as sole carbon sources. To restore growth on fructose, an alternative pathway, similar to the fructose-6-phosphate shunt of heterofermentative bifidobacteria, was established. For this, the xfp gene from Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp; Meile et al. in J Bacteriol 183:2929–2936, 2001), was expressed in R. eutropha H16 PHB⁻4 Δeda. This Xfp catalyzes the phosphorolytic cleavage of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate as well as of xylulose 5-phosphate to glyceralaldehyde 3-phosphate and acetylphosphate. The recombinant strain showed phosphoketolase (PKT) activity on either substrate, and was able to use fructose as sole carbon source for growth, because PKT is the only enzyme that is missing in R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. The Xfp-expressing strain R. eutropha H16 PHB⁻4 Δeda (pBBR1MCS-3::xfp) should be applicable for a novel variant of a plasmid addiction system to stably maintain episomally encoded genetic information during fermentative production processes. Plasmid addiction systems are often used to ensure plasmid stability in many biotechnology relevant microorganisms and processes without the need to apply external selection pressure, like the addition of antibiotics. By episomal expression of xfp in a R. eutropha H16 mutant lacking KDPG aldolase activity and cultivation in mineral salt medium with fructose as sole carbon source, the growth of this bacterium was addicted to the constructed xfp harboring plasmid. This novel selection principle extends the applicability of R. eutropha H16 as production platform in biotechnological processes.     

The H(2) sensor of Ralstonia eutropha is a member of the subclass of regulatory [NiFe] hydrogenases

Two energy-generating hydrogenases enable the aerobic hydrogen bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus) to use molecular hydrogen as the sole energy source. The complex synthesis of the nickel-iron-containing enzymes has to be efficiently regulated in response to H(2), which is available in low amounts in aerobic environments. H(2) sensing in R. eutropha is achieved by a hydrogenase-like protein which controls the hydrogenase gene expression in concert with a two-component regulatory system. In this study we show that the H(2) sensor of R. eutropha is a cytoplasmic protein. Although capable of H(2) oxidation with redox dyes as electron acceptors, the protein did not support lithoautotrophic growth in the absence of the energy-generating hydrogenases. A specifically designed overexpression system for R. eutropha provided the basis for identifying the H(2) sensor as a nickel-containing regulatory protein. The data support previous results which showed that the sensor has an active site similar to that of prototypic [NiFe] hydrogenases (A. J. Pierik, M. Schmelz, O. Lenz, B. Friedrich, and S. P. J. Albracht, FEBS Lett. 438:231-235, 1998). It is demonstrated that in addition to the enzymatic activity the regulatory function of the H(2) sensor is nickel dependent. The results suggest that H(2) sensing requires an active [NiFe] hydrogenase, leaving the question open whether only H(2) binding or subsequent H(2) oxidation and electron transfer processes are necessary for signaling. The regulatory role of the H(2)-sensing hydrogenase of R. eutropha, which has also been investigated in other hydrogen-oxidizing bacteria, is intimately correlated with a set of typical structural features. Thus, the family of H(2) sensors represents a novel subclass of [NiFe] hydrogenases denoted as the "regulatory hydrogenases."