Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans

Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and pharmaceuticals. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. Ch-MAL is a dimer in solution, consisting of two identical subunits (∼49 kDa each), and requires Mg²⁺ and K⁺ ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 70°C (k _cat = 78 s⁻¹ and K _m = 16 mM). Heat inactivation assays showed that Ch-MAL is stable at 50°C for >4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate, and propylfumarate as substrates in the ammonia addition reaction. The enzyme also processes methylamine, ethylamine, hydrazine, hydroxylamine, and methoxylamine as nucleophiles that can replace ammonia in the addition to mesaconate, resulting in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess (>98% de). This newly identified thermostable MAL appears to be a potentially attractive biocatalyst for the stereoselective synthesis of aspartic acid derivatives on large (industrial) scale.     

Purification and catalytic properties of a CO-oxidizing:H2-evolving enzyme complex from Carboxydothermus hydrogenoformans.

From the membrane fraction of the Gram-positive bacterium Carboxydothermus hydrogenoformans, an enzyme complex catalyzing the conversion of CO to CO2 and H2 was purified. The enzyme complex showed maximal CO-oxidizing:H2-evolving enzyme activity with 5% CO in the headspace (450 U per mg protein). Higher CO concentrations inhibited the hydrogenase present in the enzyme complex. For maximal activity, the enzyme complex had to be activated by either CO or strong reductants. The enzyme complex also catalyzed the CO- or H2-dependent reduction of methylviologen at 5900 and 180 U per mg protein, respectively. The complex was found to be composed of six hydrophilic and two hydrophobic polypeptides. The amino-terminal sequences of the six hydrophilic subunits were determined allowing the identification of the encoding genes in the preliminary genome sequence of C. hydrogenoformans. From the sequence analysis it was deduced that the enzyme complex is formed by a Ni-containing carbon monoxide dehydrogenase (CooS), an electron transfer protein containing four [4Fe-4S] clusters (CooF) and a membrane bound [NiFe] hydrogenase composed of four hydrophilic subunits and two membrane integral subunits. The hydrogenase part of the complex shows high sequence similarity to members of a small group of [NiFe] hydrogenases with sequence similarity to energy conserving NADH:quinone oxidoreductases. The data support a model in which the enzyme complex is composed of two catalytic sites, a CO-oxidizing site and a H2-forming site, which are connected via a different iron-sulfur cluster containing electron transfer subunits. The exergonic redox reaction catalyzed by the enzyme complex in vivo has to be coupled to energy conservation, most likely via the generation of a proton motive force.     

Novel physiological features of Carboxydothermus hydrogenoformans and Thermoterrabacterium ferrireducens

Carboxydothermus hydrogenoformans is able to grow by conversion of CO to H2 and CO2. Besides CO, only pyruvate was described as serving as an energy source. Based on 16S rRNA gene sequence similarity, C. hydrogenoformans is closely related to Thermoterrabacterium ferrireducens. T. ferrireducens is like C. hydrogenoformans a gram-positive, thermophilic, strict anaerobic bacterium. However, it is capable of using various electron donors and acceptors for growth. Growth of C. hydrogenoformans with multiple electron donors and acceptors was tested. C. hydrogenoformans oxidized formate, lactate, glycerol, CO, and H2 with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor. Sulfite, thiosulfate, sulfur, nitrate, and fumarate were reduced with lactate as an electron donor. T. ferrireducens oxidized CO with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor but did not produce H2 from CO. In contrast to what was published before, T. ferrireducens was able to grow on lactate with sulfite, sulfur, and nitrate as electron acceptors.     

Novel Physiological Features of Carboxydothermus hydrogenoformans and Thermoterrabacterium ferrireducens

Carboxydothermus hydrogenoformans is able to grow by conversion of CO to H₂ and CO₂. Besides CO, only pyruvate was described as serving as an energy source. Based on 16S rRNA gene sequence similarity, C. hydrogenoformans is closely related to Thermoterrabacterium ferrireducens. T. ferrireducens is like C. hydrogenoformans a gram-positive, thermophilic, strict anaerobic bacterium. However, it is capable of using various electron donors and acceptors for growth. Growth of C. hydrogenoformans with multiple electron donors and acceptors was tested. C. hydrogenoformans oxidized formate, lactate, glycerol, CO, and H₂ with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor. Sulfite, thiosulfate, sulfur, nitrate, and fumarate were reduced with lactate as an electron donor. T. ferrireducens oxidized CO with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor but did not produce H₂ from CO. In contrast to what was published before, T. ferrireducens was able to grow on lactate with sulfite, sulfur, and nitrate as electron acceptors.     

Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans

Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans. Both enzymes differ in their subunit molecular masses, N-terminal sequences, peptide maps, and immunological reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in association with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO + H(2)O --> CO(2) + 2 e(-) + 2 H(+). Oxidized viologen dyes are effective electron acceptors. The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) micromol of CO oxidized min(-1) mg(-1) of protein (methyl viologen, pH 8.0, 70 degrees C). The two enzymes oxidize CO very efficiently, as indicated by k(cat)/K(m) values at 70 degrees C of 1.3. 10(9) M(-1) CO s(-1) (CODH I) and 1.7. 10(9) M(-1) CO s(-1) (CODH II). The apparent K(m) values at pH 8.0 and 70 degrees C are 30 and 18 microM CO for CODH I and CODH II, respectively. Acetyl coenzyme A synthase activity is not associated with the enzymes. CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers containing 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, respectively. Electron paramagnetic resonance (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.     

Biocatalytic characterization of a short-chain alcohol dehydrogenase with broad substrate specificity from thermophilic Carboxydothermus hydrogenoformans

The gene encoding a novel short-chain alcohol dehydrogenase in the thermophilic bacterium, Carboxydothermus hydrogenoformans, was identified and overexpressed in Escherichia coli. The enzyme was thermally stable and displayed the highest activity at 70 °C and pH 6.0. It preferred NAD(H) over NADP(H) as a cofactor and exhibited broad substrate specificity towards aliphatic ketones, cycloalkanones, aromatic ketones, and ketoesters. Furthermore, ethyl benzoylformate was asymmetrically reduced by the purified enzyme, using an additional coupled NADH regeneration system, with 95 % conversion and in an enantiomeric excess of (99.9 %). The results of this study may lead to the discovery of a novel method for asymmetric reduction of alcohols, which is an important tool in organic synthesis.     

Genetic analysis of Carboxydothermus hydrogenoformans carbon monoxide dehydrogenase genes cooF and cooS

Carboxydothermus hydrogenoformans is an extremely thermophilic, Gram-positive bacterium growing on carbon monoxide (CO) as single carbon and energy source and producing only H(2) and CO(2). Carbon monoxide dehydrogenase is a key enzyme for CO metabolism. The carbon monoxide dehydrogenase genes cooF and cooS from C. hydrogenoformans were cloned and sequenced. These genes showed the highest similarity to the cooF genes from the archaeon Archaeoglobus fulgidus and the cooS gene from the bacterium Rhodospirillum rubrum, respectively. The cooS gene was identified immediately downstream of cooF, however, the cooF and cooS genes from C. hydrogenoformans have substantially different codon usage, and the cooF gene Arg codon usage pattern, dominated by AGA and AGG, resembles the archaeal pattern. The data therefore suggest lateral transfer of these genes, possibly from different donor species.     

A Simple,Large-Scale Overexpression Method of Deriving Carbon Monoxide Dehydrogenase II from Thermophilic Bacterium Carboxydothermus hydrogenoformans

We established an Na₂S-free, large-scale overexpression system of deriving CODH II from thermophilic bacterium Carboxydothermus hydrogenoformans in Escherichia coli using a large-scale fermentor. Recombinant-CODH II showed a CO oxidation activity of 9,600 U/mg. In addition, recombinant-CODH II exhibited considerable CO₂ reduction activity, of 16.9 U/mg.     

A simple, large-scale overexpression method of deriving carbon monoxide dehydrogenase II from thermophilic bacterium Carboxydothermus hydrogenoformans

We established an Na(2)S-free, large-scale overexpression system of deriving CODH II from thermophilic bacterium Carboxydothermus hydrogenoformans in Escherichia coli using a large-scale fermentor. Recombinant-CODH II showed a CO oxidation activity of 9,600 U/mg. In addition, recombinant-CODH II exhibited considerable CO(2) reduction activity, of 16.9 U/mg.     

Computational kinetic studies of pyruvate metabolism in Carboxydothermus hydrogenoformans Z-2901 for improved hydrogen production

Hydrogen is considered as a renewable energy source and it is also regarded as future fuel. Currently, hydrogen production through a biotechnological approach is a research priority. Hydrogenogens, a microbial species, are of significant interest to researchers because of their ability to produce biological hydrogen. Carboxydothermus hydrogenoformans Z-2901 is one among the hydrogenogens that can grow anaerobically by utilizing pyruvate as a carbon source, and can produce molecular hydrogen. In the present study, we performed an in silico kinetic simulation using the available Kyoto Encyclopedia of Genes and Genomes (KEGG) model and reconstructed pyruvate metabolism in C. hydrogenoformans Z-2901. During this metabolism, dissimilation of pyruvate leads to the formation of energy co-factors, such as ATP and NAD⁺/ NADH, and the level of these co-factors influences the specific growth rate of organism and hydrogen production. Our strategy for improving hydrogen production involves maximizing the ATP and NAD⁺ yield by modification of kinetic properties and adding new reactions in pyruvate metabolism through metabolic pathway reconstruction. Moreover, the influence of phosphoenol pyruvate carboxylase and pyruvate dehydrogenase enzyme concentration on cofactor productions was also simulated. The theoretical molar yield of ATP and NAD⁺ were obtained as 2.32 and 1.83 mM, respectively, from 1 mM/mg of phosphoenol pyruvate (PEP) utilization. A higher yield of ATP is achieved when the PEP level reaches 5 mM/mg. This work also suggests that PEP can be considered as an alternative substrate. In conclusion, the simulation results reported in this paper can be applied to design and evaluate strategies of strain construction for optimal hydrogen yield in C. hydrogenoformans.     

Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans Z-2901

We report here the sequencing and analysis of the genome of the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. This species is a model for studies of hydrogenogens, which are diverse bacteria and archaea that grow anaerobically utilizing carbon monoxide (CO) as their sole carbon source and water as an electron acceptor, producing carbon dioxide and hydrogen as waste products. Organisms that make use of CO do so through carbon monoxide dehydrogenase complexes. Remarkably, analysis of the genome of C. hydrogenoformans reveals the presence of at least five highly differentiated anaerobic carbon monoxide dehydrogenase complexes, which may in part explain how this species is able to grow so much more rapidly on CO than many other species. Analysis of the genome also has provided many general insights into the metabolism of this organism which should make it easier to use it as a source of biologically produced hydrogen gas. One surprising finding is the presence of many genes previously found only in sporulating species in the Firmicutes Phylum. Although this species is also a Firmicutes, it was not known to sporulate previously. Here we show that it does sporulate and because it is missing many of the genes involved in sporulation in other species, this organism may serve as a “minimal” model for sporulation studies. In addition, using phylogenetic profile analysis, we have identified many uncharacterized gene families found in all known sporulating Firmicutes, but not in any non-sporulating bacteria, including a sigma factor not known to be involved in sporulation previously.     

CO-induced structural rearrangement of the C cluster in Carboxydothermus hydrogenoformans CO dehydrogenase-evidence from Ni K-edge X-ray absorption spectroscopy

We have examined the C cluster in type II CO dehydrogenase (CODH) from Carboxydothermus hydrogenformans using Ni K-edge X-ray absorption near edge spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy. The enzyme was studied under three conditions: "as-isolated" and after treatment with CO or Ti(III). The shape of the Ni K-edge changes slightly between the different conditions, but no significant edge shift is seen, suggesting that the C cluster contains Ni(II) in both forms. The Ni EXAFS of as-isolated CODH can be simulated with 4 Ni-S interactions at 2.20 A with a large spread in distances. A light atom (C, N, O) is not required to fit the spectrum. After CO treatment, significant changes are observed in the EXAFS. A new feature appears at approximately 2.7 A; this component is consistent with a Ni-Fe interaction. The average Ni-S distance also expands to approximately 2.25 A. The changes between the two forms suggest that the active site (C cluster) undergoes structural rearrangement after CO treatment, and the observed changes help reconcile the two different crystal structures. The implications of the structural change for the enzyme activation and mechanism are discussed.     

Carbon monoxide conversion by thermophilic sulfate-reducing bacteria in pure culture and in co-culture with Carboxydothermus hydrogenoformans

Biological sulfate (SO₄) reduction with carbon monoxide (CO) as electron donor was investigated. Four thermophilic SO₄-reducing bacteria, Desulfotomaculum thermoacetoxidans (DSM 5813), Thermodesulfovibrio yellowstonii (ATCC 51303), Desulfotomaculum kuznetsovii (DSM 6115; VKM B-1805), and Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum (DSM 14055), were studied in pure culture and in co-culture with the thermophilic carboxydotrophic bacterium Carboxydothermus hydrogenoformans (DSM 6008). D. thermoacetoxidans and T. yellowstonii were extremely sensitive to CO: their growth on pyruvate was completely inhibited at CO concentrations above 2% in the gas phase. D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were less sensitive to CO. In pure culture, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were able to grow on CO as the only electron donor and, in particular in the presence of hydrogen/carbon dioxide, at CO concentrations as high as 50–70%. The latter SO₄ reducers coupled CO oxidation to SO₄ reduction, but a large part of the CO was converted to acetate. In co-culture with C. hydrogenoformans, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum could even grow with 100% CO (P_CO=120 kPa).     

Interaction of potassium cyanide with the [Ni-4Fe-5S] active site cluster of CO dehydrogenase from Carboxydothermus hydrogenoformans

The Ni-Fe carbon monoxide (CO) dehydrogenase II (CODHII(Ch)) from the anaerobic CO-utilizing hydrogenogenic bacterium Carboxydothermus hydrogenoformans catalyzes the oxidation of CO, presumably at the Ni-(micro(2)S)-Fe1 subsite of the [Ni-4S-5S] cluster in the active site. The CO oxidation mechanism proposed on the basis of several CODHII(Ch) crystal structures involved the apical binding of CO at the nickel ion and the activation of water at the Fe1 ion of the cluster. To understand how CO interacts with the active site, we have studied the reactivity of the cluster with potassium cyanide and analyzed the resulting type of nickel coordination by x-ray absorption spectroscopy. Cyanide acts as a competitive inhibitor of reduced CODHII(Ch) with respect to the substrate CO and is therefore expected to mimic the substrate. It inhibits the enzyme reversibly, forming a nickel cyanide. In this reaction, one of the four square-planar sulfur ligands of nickel is replaced by the carbon atom of cyanide, suggesting removal of the micro(2)S from the Ni-(micro(2)S)-Fe1 subsite. Upon reactivation of the inhibited enzyme, cyanide is released, and the square-planar coordination of nickel by 4S ligands is recovered, which includes the reformation of the Ni-(micro(2)S)-Fe1 bridge. The results are summarized in a model of the CO oxidation mechanism at the [Ni-4Fe-5S] active site cluster of CODHII(Ch) from C. hydrogenoformans.     

A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans

The H(+) proton-translocating inorganic pyrophosphatase (H(+)-PPase) family is composed of two phylogenetically distinct types of enzymes: K(+)-dependent and K(+)-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H(+)-PPase from the thermophilic bacterium Carboxydothermus hydrogenoformans. Both PP(i)-hydrolyzing and PP(i)-energized H(+) translocation activities of the recombinant enzyme in Escherichia coli inner membrane vesicles are strictly K(+)-dependent. Here we deduce the K(+) requirement of all available H(+)-PPase sequences based on the K(+) dependence of C. hydrogenoformans H(+)-PPase in conjunction with phylogenetic analyses. Our data reveal that K(+)-independent H(+)-PPases possess conserved Lys and Thr that are absent in K(+)-dependent H(+)-PPases. We further demonstrate that a A460K substitution in C. hydrogenoformans H(+)-PPase is sufficient to confer K(+) independence to both PP(i) hydrolysis and PP(i)-energized H(+) translocation. In contrast, a A463T mutation does not affect the K(+) dependence of H(+)-PPase.     

Characterization of a soluble oxidoreductase from the thermophilic bacterium Carboxydothermus ferrireducens

An NAD(P)H-dependent oxidoreductase has been purified approximately 40-fold from the soluble protein fraction of the dissimilatory iron-reducing, anaerobic, thermophilic bacterium Carboxydothermus ferrireducens. The enzyme, a flavoprotein, has broad-substrate specificity—reducing Fe³⁺, Cr⁶⁺, and AQDS with rates of 0.31, 0.33, and 3.3 U mg⁻¹ protein and calculated NADH oxidation turnover numbers of 0.25, 0.25, and 2.5 s⁻¹, respectively. Numerous quinones are reduced via a two-electron transfer from NAD(P)H to quinone, thus participating in managing oxidative stress by avoiding the formation of semiquinone radicals.     

Spectroscopic properties of a novel neutral proteinase from Saccharomonospora canescens

Three distinct DNA polymerase fractions (A, B and C), were isolated from Trypanosoma cruzi epimastigote forms. Fraction A is a low molecular mass enzyme corresponding to beta-like DNA polymerase of T. cruzi. Fraction B co-purified along several purification steps with fraction A, but in the last step it was clearly separated by a phosphocellulose chromatography. Fraction C was separated from fractions A and B by binding to DEAE-cellulose column, since the other two fractions were eluted in the flowthrough. This enzyme has an apparent native molecular mass of 100 kDa and showed a high preference for poly(dC)-oligo(dG) among different template-primers tested as substrate. Western-blot and biochemical analysis strongly suggest that the three DNA polymerase fractions correspond to different molecular entities. These results are in agreement with the idea that fraction C is a new DNA polymerase of T. cruzi, not described before.     

Spectroscopic properties of the main-form and high-salt peridinin-chlorophyll a proteins from Amphidinium carterae

The main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a proteins from the dinoflagellate Amphidinium carterae were investigated using absorption, fluorescence, fluorescence excitation, two-photon, and fast-transient optical spectroscopy. Pigment analysis has demonstrated previously that MFPCP contains eight peridinins and two chlorophyll (Chl) a molecules, whereas HSPCP has six peridinins and two Chl a molecules [Sharples, F. P., et al. (1996) Biochim. Biophys. Acta 1276, 117-123]. Absorption spectra of the complexes were recorded at 10 K and analyzed in the 400-600 nm region by summing the individual 10 K spectra of Chl a and peridinin recorded in 2-MTHF. The absorption spectral profiles of the complexes in the Q(y) region between 650 and 700 nm were fit using Gaussian functions. The absorption and fluorescence spectra from both complexes exhibit several distinguishing features that become evident only at cryogenic temperatures. In particular, at low temperatures the Q(y) transitions of the Chls bound in the HSPCP complex are split into two well-resolved bands. Fluorescence excitation spectroscopy has revealed that the peridinin-to-Chl a energy transfer efficiency is high (>95%). Transient absorption spectroscopy has been used to measure the rate of energy transfer between the two bound Chls which is a factor of 2.9 slower in HSPCP than in MFPCP. The kinetic data are interpreted in terms of the F?rster mechanism describing energy transfer between weakly coupled, spatially fixed, donor-acceptor Chl a molecules. The study provides insight into the molecular factors that control energy transfer in this class of light-harvesting pigment-protein complexes.