A universal polyphosphate kinase: PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate.

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the ß-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP. KEY POINTS: • PPK2c of R. eutropha can be used for regeneration of any NTP or dNTP. • PPK2c is highly unspecific and accepts all purine and pyrimidine nucleotides. • PPK2c forms polyphosphate granules in vitro from any NTP.

Expanded amino acid sequence of the PhaC box in the active center of polyhydroxyalkanoate synthases.

Polyhydroxyalkanoate (PHA) synthases catalyze the polymerization reaction of the acyl moiety of hydroxyacyl-coenzyme A into polyester. The catalytic subunit PhaC of PHA synthase has the PhaC box sequence at the active site that is typically described as G-X-C-X-G-G (X is an arbitrary amino acid), and cysteine is an active center. In this study, an amino acid replacement was introduced into the PhaC box of the PHA synthase derived from Ralstonia eutropha (PhaCRe ) to investigate the importance of highly conserved residues in polymerizing activity. Point mutagenesis revealed that PhaCRe mutants with the expanded PhaC box sequence ([GAST]-X-C-X-[GASV]-[GA]) are functional PHA synthases. These findings highlight the low mutational robustness of the last glycine residue in the PhaC box as well as that of the active center cysteine.

Synthesis of Formate from CO2 Gas Catalyzed by an O2-Tolerant NAD-Dependent Formate Dehydrogenase and Glucose Dehydrogenase.

Direct biocatalytic conversion of CO2 to formic acid is an attractive means of reversibly storing energy in chemical bonds. Formate dehydrogenases (FDHs) are a heterogeneous group of enzymes that catalyze the oxidation of formic acid to carbon dioxide, generating two protons and two electrons. Several FDHs have recently been reported to catalyze the reverse reaction, i.e., the reduction of carbon dioxide to formic acid, under appropriate conditions. The main challenges with these enzymes are relatively low rates of CO2 reduction and high oxygen sensitivity. Our earlier studies (Yu et al. (2017) J. Biol. Chem. 292, 16872-16879) have shown that the FdsABG formate dehydrogenase from Cupriavidus necator is able to effectively catalyze the reduction of CO2, using NADH as a source of reducing equivalents, with a good oxygen tolerance. On the basis of this result, we have developed a highly thermodynamically efficient and cost-effective biocatalytic process for the transformation of CO2 to formic acid using FdsABG. We have  cloned the full-length soluble formate dehydrogenase (FdsABG) from C. necator and expressed it in Escherichia coli with a His-tag fused to the N terminus of the FdsG subunit; this overexpression system has greatly simplified the FdsABG purification process. Importantly, we have also combined this recombinant C. necator FdsABG with another enzyme, glucose dehydrogenase, for continuous regeneration of NADH for CO2 reduction and demonstrated that the combined system is highly effective in reducing CO2 to formate. The results indicate that this system shows significant promise for the future development of an enzyme-based system for the industrial reduction of CO2.

PHA synthase (PhaC): interpreting the functions of bioplastic-producing enzyme from a structural perspective.

Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.

Studies on the aerobic utilization of synthesis gas (syngas) by wild type and recombinant strains of Ralstonia eutropha H16.

The biotechnical platform strain Ralstonia eutropha H16 was genetically engineered to express a cox subcluster of the carboxydotrophic Oligotropha carboxidovoransOM5, including (i) the structural genes coxM, -S and -L, coding for an aerobic carbon monoxide dehydrogenase (CODH) and (ii) the genes coxD, -E, -F and -G, essential for the maturation of CODH. The coxOc genes expressed under control of the CO2 -inducible promoter PL enabled R. eutropha to oxidize CO to CO2 for the use as carbon source, as demonstrated by 13 CO experiments, but the recombinant strains remained dependent on H2 as external energy supply. Therefore, a synthetic metabolism, which could be described as 'carboxyhydrogenotrophic', was established in R. eutropha. With this extension of the bacterium's substrate range, growth in CO-, H2 - and CO2 -containing artificial synthesis gas atmosphere was enhanced, and poly(3-hydroxybutyrate) synthesis was increased by more than 20%.

Conversion of cysteine to 3-mercaptopyruvic acid by bacterial aminotransferases.

3-Mercaptopyruvate (3MPy), a structural analog of 3-mercaptopropionic acid, is a precursor compound for biosynthesis of polythioesters in bacteria. The cost-effectiveness and sustainability of the whole process could be greatly improved by using the cysteine degradation pathway for an intracellular supply of 3MPy. Transamination of cysteine to its corresponding α-keto acid 3MPy is catalyzed by cysteine aminotransferases (CAT). However, CAT activity has so far not been described for bacterial aminotransferases (AT), and it was unknown whether they can be applied for the conversion of cysteine to 3MPy. In this study, we selected eight bacterial aminotransferases based on sequence homology to CAT of Rattus norvegicus (Got1). The aminotransferases included four aspartate aminotransferases (AATs) and four aromatic amino acid aminotransferases (ArATs) from Advenella mimigardefordensis DPN7, Escherichia coli MG1655, Shimwellia blattae ATCC 33430, Ralstonia eutropha H16 and Paracoccus denitrificans PD1222. For a more detailed characterization, all selected AAT or ArAT encoding genes were heterologously expressed in E. coli and purified. CAT activity was detected for all aminotransferases when a novel continuous coupled enzyme assay was applied. Kinetic studies revealed the highest catalytic efficiency of 5.1mM/s for AAT from A. mimigardefordensis. Formation of 3MPy from cysteine could additionally be verified by an optimized approach using derivatization of 3MPy with the Girard T reagent and liquid chromatography-mass spectrometry analyses.

Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16.

Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3'-thiodipropionic acid (TDP) and 3,3'-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs.

Insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions.

The polyhydroxyalkanoate (PHA) synthase catalyzes the synthesis of PHA and remains attached to the hydrophobic PHA inclusions it creates. Although this feature is actively exploited to generate functionalized biobeads via protein engineering, little is known about the structure of the PHA synthase. Here, the surface topology of Ralstonia eutropha PHA synthase was probed to inform rational protein engineering toward the production of functionalized PHA beads. Surface-exposed residues were detected by conjugating biotin to inclusion-bound PHA synthase and identifying the biotin-conjugated lysine and cysteine residues using peptide fingerprinting analysis. The identified sites (K77, K90, K139, C382, C459, and K518) were investigated as insertion sites for the generation of new protein fusions. Insertions of FLAG epitopes into exposed sites K77, K90, K139, and K518 were tolerated, retaining >65 % of in vivo activity. Sites K90, K139, and K518 were also tested by insertion of the immunoglobulin G (IgG)-binding domain (ZZ), successfully producing PHA inclusions able to bind human IgG in vitro. Although simultaneous insertions of the ZZ domain into two sites was permissive, insertion at all three lysine sites inactivated the synthase. The K90/K139 double ZZ insertion had the optimum IgG-binding capacity of 16 mg IgG/g wet PHA beads and could selectively purify the IgG fraction from human serum. Overall, this study identified surface-exposed flexible regions of the PHA synthase which either tolerate protein/peptide insertions or are critical for protein function. This further elucidates the structure and function of PHA synthase and provides new opportunities for generating functionalized PHA biobeads.

Impact of the iron-sulfur cluster proximal to the active site on the catalytic function of an O2-tolerant NAD(+)-reducing [NiFe]-hydrogenase.

The soluble NAD(+)-reducing hydrogenase (SH) from Ralstonia eutropha H16 belongs to the O2-tolerant subtype of pyridine nucleotide-dependent [NiFe]-hydrogenases. To identify molecular determinants for the O2 tolerance of this enzyme, we introduced single amino acids exchanges in the SH small hydrogenase subunit. The resulting mutant strains and proteins were investigated with respect to their physiological, biochemical, and spectroscopic properties. Replacement of the four invariant conserved cysteine residues, Cys41, Cys44, Cys113, and Cys179, led to unstable protein, strongly supporting their involvement in the coordination of the iron-sulfur cluster proximal to the catalytic [NiFe] center. The Cys41Ser exchange, however, resulted in an SH variant that displayed up to 10% of wild-type activity, suggesting that the coordinating role of Cys41 might be partly substituted by the nearby Cys39 residue, which is present only in O2-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O2 sensitivity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O2-tolerant SH representatives did not greatly affect the H2-oxidizing activity in the presence of O2. Remarkably, all mutant enzymes investigated by electron paramagnetic resonance spectroscopy did not reveal significant spectral changes in relation to wild-type SH, showing that the proximal iron-sulfur cluster does not contribute to the wild-type spectrum. Interestingly, exchange of Trp42 by serine resulted in a completely redox-inactive [NiFe] site, as revealed by infrared spectroscopy and H2/D(+) exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed.

Crystal structure and biochemical properties of the (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha.

3-Hydroxybutyryl-CoA dehydrogenase is an enzyme involved in the synthesis of the biofuel n-butanol by converting acetoacetyl-CoA to 3-hydroxybutyryl-CoA. To investigate the molecular mechanism of n-butanol biosynthesis, we determined crystal structures of the Ralstonia eutropha-derived 3-hydroxybutyryl-CoA dehydrogenase (RePaaH1) in complex with either its cofactor NAD(+) or its substrate acetoacetyl-CoA. While the biologically active structure is dimeric, the monomer of RePaaH1 comprises two separated domains with an N-terminal Rossmann fold and a C-terminal helical bundle for dimerization. In this study, we show that the cofactor-binding site is located on the Rossmann fold and is surrounded by five loops and one helix. The binding mode of the acetoacetyl-CoA substrate was found to be that the adenosine diphosphate moiety is not highly stabilized compared with the remainder of the molecule. Residues involved in catalysis and substrate binding were further confirmed by site-directed mutagenesis experiments, and kinetic properties of RePaaH1were examined as well. Our findings contribute to the understanding of 3-hydroxybutyryl-CoA dehydrogenase catalysis, and will be useful in enhancing the efficiency of n-butanol biosynthesis by structure based protein engineering.

Modification of ß-oxidation pathway in Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from soybean oil.

Ralstonia eutropha H16 is a useful platform for metabolic engineering aiming at efficient production of polyhydroxyalkanaotes being attracted as practical bioplastics. This study focused on bifunctional (S)-specific 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase encoded by fadB to obtain information regarding ß-oxidation in this bacterium and to achieve compositional regulation of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] synthesized from soybean oil. In addition to two FadB homologs (FadB1 and FadB') encoded within the previously identified ß-oxidation gene clusters on the chromosome 1, a gene of third homolog (FadB2) was found on chromosome 2 of R. eutropha. The fadB homologs were disrupted in R. eutropha strain NSDG expressing a mutant gene of PHA synthase from Aeromonas caviae. The gene disruptions affected neither growth nor PHA production on fructose. On soybean oil, fadB' deletion led to reduction of PHA quantity attributed to decrease of 3HB unit, while fadB1 deletion slightly increased 3HHx composition without serious negative impact on both cell growth and PHA biosynthesis. Double deletion of fadB1 and fadB' significantly impaired the cell growth and PHA biosynthesis, indicating the major roles of fadB1 and fadB' in ß-oxidation. When fadB1 was deleted in several engineered strains of R. eutropha possessing additional (R)-enoyl-CoA hydratase gene(s), the net amounts of 3HHx unit in the PHA fractions showed 6-21% increase probably due to slightly enhanced supply of medium-chain-length 2-enoyl-CoAs through the partially impaired ß-oxidation. These results demonstrated that modification of ß-oxidation by fadB1 deletion was effective for increasing 3HHx composition in the copolyesters produced from soybean oil.

Redox-dependent structural transformations of the [4Fe-3S] proximal cluster in O2-tolerant membrane-bound [NiFe]-hydrogenase: a DFT study.

Broken-symmetry density functional theory (BS-DFT) has been used to address the redox-dependent structural changes of the proximal [4Fe-3S] cluster, implicated in the O2-tolerance of membrane-bound [NiFe]-hydrogenase (MBH). The recently determined structures of the [4Fe-3S] cluster together with its protein ligands were studied at the reduced [4Fe-3S](3+), oxidized [4Fe-3S](4+), and superoxidized [4Fe-3S](5+) levels in context of their relative energies and protonation states. The observed proximal cluster conformational switch, concomitant with the proton transfer from the cysteine Cys20 backbone amide to the nearby glutamate Glu76 carboxylate, is found to be a single-step process requiring ~12-17 kcal/mol activation energy at the superoxidized [4Fe-3S](5+) level. At the more reduced [4Fe-3S](4+/3+) oxidation levels, this rearrangement has at least 5 kcal/mol higher activation barriers and prohibitively unfavorable product energies. The reverse transformation of the proximal cluster is a fast unidirectional process with ~8 kcal/mol activation energy, triggered by one-electron reduction of the superoxidized species. A previously discussed ambiguity of the Glu76 carboxylate and 'special' Fe4 iron positions in the superoxidized cluster is now rationalized as a superposition of two local minima, where Glu76-Fe4 coordination is either present or absent. The calculated 12.3-17.9 MHz (14)N hyperfine coupling (HFC) for the Fe4-bound Cys20 backbone nitrogen is in good agreement with the large 13.0/14.6 MHz (14)N couplings from the latest HYSCORE/ENDOR studies.

Formation of [Ni(III)(κ(1)-S2CH)(P(o-C6H3-3-SiMe3-2-S)3)]- via CS2 insertion into nickel(III) hydride containing [Ni(III)(H)(P(o-C6H3-3-SiMe3-2-S)3)]-.

Insertion of CS2 into the thermally unstable nickel(III) hydride [PPN][Ni(H)(P(o-C6H3-3-SiMe3-2-S)3)] (1), freshly prepared from the reaction of [PPN][Ni(OC6H5)P(C6H3-3-SiMe3-2-S)3] and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (HBpin; pin = OCMe2CMe2O) in tetrahydrofuran at -80 °C via a metathesis reaction, readily affords [PPN][Ni(III)(κ(1)-S2CH)(P(o-C6H3-3-SiMe3-2-S)3)] (2) featuring a κ(1)-S2CH moiety.

New insights into activation and substrate recognition of polyhydroxyalkanoate synthase from Ralstonia eutropha.

The polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) shows a lag time for the start of its polymerization reaction, which complicates kinetic analysis of PhaC(Re). In this study, we found that the lag can be virtually eliminated by addition of 50 mg/L TritonX-100 detergent into the reaction mixture, as well as addition of 2.5 g/L Hecameg detergent as previously reported by Gerngross and Martin (Proc Natl Sci USA 92: 6279-6283, 1995). TritonX-100 is an effective lag eliminator working at much lower concentration than Hecameg. Kinetic analysis of PhaC(Re) was conducted in the presence of TritonX-100, and PhaC(Re) obeyed Michaelis-Menten kinetics for (R)-3-hydroxybutyryl-CoA substrate. In inhibitory assays using various compounds such as adenosine derivatives and CoA derivatives, CoA free acid showed competitive inhibition but other compounds including 3'-dephospho CoA had no inhibitory effect. Furthermore, PhaC(Re) showed a considerably reduced reaction rate for 3'-dephospho (R)-3-hydroxybutyryl CoA substrate and did not follow typical Michaelis-Menten kinetics. These results suggest that the 3'-phosphate group of CoA plays a critical role in substrate recognition by PhaC(Re).

(R)-Cysteate-nitrogen assimilation by Cupriavidus necator H16 with excretion of 3-sulfolactate: a patchwork pathway.

Cupriavidus necator H16 grew exponentially with (R)-cysteate, a structural analogue of aspartate, as sole source of nitrogen in succinate-salts medium. Utilization of cysteate was quantitative and concomitant with growth and with the excretion of the deaminated product (R)-sulfolactate, which was identified thoroughly. The deaminative pathway started with transport of (R)-cysteate into the cell, which we attributed to an aspartate transporter. Transamination to sulfopyruvate involved an aspartate/(R)-cysteate:2-oxoglutarate aminotransferase (Aoa/Coa) and regeneration of the amino group acceptor by NADP⁺-coupled glutamate dehydrogenase. Reduction of sulfopyruvate to (R)-sulfolactate was catalyzed by a (S)-malate/(R)-sulfolactate dehydrogenase (Mdh/Sdh). Excretion of the sulfolactate could be attributed to the sulfite/organosulfonate exporter TauE, which was co-encoded and co-expressed, with sulfoacetaldehyde acetyltransferase (Xsc), though Xsc was irrelevant to the current pathway. The metabolic enzymes could be assayed biochemically. Aoa/Coa and Mdh/Sdh were highly enriched by protein separation, partly characterized, and the relevant locus-tags identified by peptide-mass fingerprinting. Finally, RT-PCR was used to confirm the transcription of all appropriate genes. We thus demonstrated that Cupriavidus necator H16 uses a patchwork pathway by recruitment of 'housekeeping' genes and sulfoacetaldehyde-degradative genes to scavenge for (R)-cysteate-nitrogen.

Reduction of benzoquinones to hydroquinones via spontaneous reaction with glutathione and enzymatic reaction by S-glutathionyl-hydroquinone reductases.

S-Glutathionyl-hydroquinone reductases (GS-HQRs) are a new class of glutathione transferases, widely present in bacteria, halobacteria, fungi, and plants. They catalyze glutathione (GSH)-dependent reduction of GS-trichloro-p-hydroquinone to trichloro-p-hydroquinone. Since GS-trichloro-p-hydroquinone is uncommon in nature, the extensive presence of GS-HQRs suggests they use common GS-hydroquinones. Here we demonstrate that several benzoquinones spontaneously reacted with GSH to form GS-hydroquinones via Michael addition, and four GS-HQRs from yeast and bacteria reduced the GS-hydroquinones to the corresponding hydroquinones. The spontaneous and enzymatic reactions led to the reduction of benzoquinones to hydroquinones with the concomitant oxidation of GSH to oxidized glutathione (GS-SG). The enzymes did not use GS-benzoquinones or other thiol-hydroquinones, for example, S-cysteinyl-hydroquinone, as substrates. Apparent kinetic parameters showed the enzymes preferred hydrophobic, bulky substrates, such as GS-menadiol. The broad substrate range and their wide distribution suggest two potential physiological roles: channeling GS-hydroquinones back to hydroquinones and reducing benzoquinones via spontaneous formation of GS-hydroquinones and then enzymatic reduction to hydroquinones. The functions are likely important in metabolic pathways with quinone intermediates.

Physiological conditions conducive to high cell density and high cyanophycin content in Ralstonia eutropha strain H16 possessing a KDPG aldolase gene-dependent addiction system.

The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.

The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre.

Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H(2) into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O(2) are considered to be central to H(2)-based technologies, such as enzymatic fuel cells and for light-driven H(2) production. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O(2) has remained unclear. Here we present the crystal structure of an O(2)-tolerant [NiFe]-hydrogenase from the aerobic H(2) oxidizer Ralstonia eutropha H16 at 1.5 Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H(2)-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H(2) oxidation and as an electron-delivering device upon O(2) attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials. The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H(2)-converting catalysts that are capable of cycling H(2) in air.

[NiFe] and [FeS] cofactors in the membrane-bound hydrogenase of Ralstonia eutropha investigated by X-ray absorption spectroscopy: insights into O(2)-tolerant H(2) cleavage.

Molecular features that allow certain [NiFe] hydrogenases to catalyze the conversion of molecular hydrogen (H(2)) in the presence of dioxygen (O(2)) were investigated. Using X-ray absorption spectroscopy (XAS), we compared the [NiFe] active site and FeS clusters in the O(2)-tolerant membrane-bound hydrogenase (MBH) of Ralstonia eutropha and the O(2)-sensitive periplasmic hydrogenase (PH) of Desulfovibrio gigas. Fe-XAS indicated an unusual complement of iron-sulfur centers in the MBH, likely based on a specific structure of the FeS cluster proximal to the active site. This cluster is a [4Fe4S] cubane in PH. For MBH, it comprises less than ~2.7 Å Fe-Fe distances and additional longer vectors of ≥3.4 Å, consistent with an Fe trimer with a more isolated Fe ion. Ni-XAS indicated a similar architecture of the [NiFe] site in MBH and PH, featuring Ni coordination by four thiolates of conserved cysteines, i.e., in the fully reduced state (Ni-SR). For oxidized states, short Ni-µO bonds due to Ni-Fe bridging oxygen species were detected in the Ni-B state of the MBH and in the Ni-A state of the PH. Furthermore, a bridging sulfenate (CysSO) is suggested for an inactive state (Ni(ia)-S) of the MBH. We propose that the O(2) tolerance of the MBH is mainly based on a dedicated electron donation from a modified proximal FeS cluster to the active site, which may favor formation of the rapidly reactivated Ni-B state instead of the slowly reactivated Ni-A state. Thereby, the catalytic activity of the MBH is facilitated in the presence of both H(2) and O(2).