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Biomedical subjects

K L Kovacs

Publications and source records attributed to K L Kovacs.

11 recordsLinked to original sources
PubMed1998

Unusual organization of the genes coding for HydSL, the stable [NiFe]hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina BBS.

The characterization of a hyd gene cluster encoding the stable, bidirectional [NiFe]hydrogenase 1 enzyme in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium belonging to the family Chromatiaceae, is presented. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K. L. Kovacs et al., J. Biol. Chem. 266:947-951, 1991; C. Bagyinka et al., J. Am. Chem. Soc. 115:3567-3585, 1993). As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes hydS and hydL, which encode the small and the large subunits, respectively. This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2. ISP1 and ISP2 are homologous to ORF5 and ORF6 in the hmc operon, coding for a transmembrane electron transfer complex in Desulfovibrio vulgaris. Other accessory proteins are not found immediately downstream or upstream of hydSL. A hup gene cluster coding for a typical hydrogen uptake [NiFe]hydrogenase in T. roseopersicina was reported earlier (A. Colbeau et al. Gene 140:25-31, 1994). The deduced amino acid sequences of the two small (hupS and hydS) and large subunit (hupL and hydL) sequences share 46 and 58% identity, respectively. The hup and hyd genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome. Both hydrogenases are associated with the photosynthetic membrane. A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source. The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by hydSL, and the inducible and unstable second hydrogenase may be the product of the hup gene cluster.

Amino Acid Sequence↗
PubMed1995

Heat-tolerant methanotrophic bacteria from the hot water effluent of a natural gas field.

Methanotrophic bacteria were isolated from a natural environment potentially favorable to heat-tolerant methanotrophs. An improved colony plate assay was developed and used to identify putative methanotrophic colonies with high confidence. Fourteen new isolates were purified and partially characterized. These new isolates exhibit a DNA sequence homology of up to 97% with the conserved regions in the mmoX and mmoC genes of the soluble methane monooxygenase (MMO)-coding gene cluster of Methylococcus capsulatus Bath. The copper regulation of soluble MMO expression in the same isolates, however, differs from that of M. capsulatus Bath, as the new isolates can tolerate up to 0.8 microM copper without loss of MMO activity while a drastic reduction of MMO activity occurs already at 0.1 microM copper in M. capsulatus Bath. The isolates can be cultivated and utilized at elevated temperatures, and their copper- and heat-tolerant MMO activity makes these bacteria ideal candidates for future biotechnological use.

Amino Acid Sequence↗
PubMed1994

Cloning and sequence of the structural (hupSLC) and accessory (hupDHI) genes for hydrogenase biosynthesis in Thiocapsa roseopersicina.

The first molecular biology study on the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is reported, namely, the construction of cosmid libraries and isolation of a hydrogenase gene cluster by hybridization with hydrogenase structural genes from the purple non-sulfur bacterium, Rhodobacter capsulatus. The sequenced gene cluster contains six open reading frames, the products of which show significant degrees of identity (from 40 to 78%) with hydrogenase gene products necessary for biosynthesis of the group-I of [NiFe]hydrogenases. The structural hupSLC genes encode the small and large hydrogenase subunits and a hydrophobic protein shown to accept electrons from hydrogenase in R. capsulatus. They are followed downstream by three genes, hupDHI, which are similar to hydrogenase accessory genes found in other bacteria.

Amino Acid Sequence↗
PubMed1994

Methane utilizing bacteria and their biotechnological applications.

Methanotroph microorganisms oxidize methane in four steps, producing methanol, formaldehyde, formate intermediers and eventually degrade methane to carbon dioxide and water. It is possible to separate the pathway into four steps in the cell free extract or after partial purification of the various enzymes. The key enzyme is a metalloenzyme, methane monooxygenase (MMO) which catalyses the oxidation of methane to methanol. MMO is also capable of biodegrading exceptionally harmful and stable chlorinated hydrocarbons. Produced by various industrial activities, most chlorinated hydrocarbons are toxic, potential and/or proven carcinogens and their decomposition challenges water treatment technologies.

Biotechnology↗
PubMed1989

Metal composition analysis of hydrogenase from Thiocapsa roseopersicina by proton induced X-ray emission spectroscopy.

Polyacrylamide gel electrophoresis combined with proton induced X-ray emission spectroscopy is suitable to identify and to determine the relative amounts of protein bound metals in situ. An analysis of the hydrogenase from Thiocapsa roseopersicina has shown the feasibility of the technique and provides new insight into the relative amount as well as the intramolecular location of Fe and Ni metal atoms in this enzyme.

Bacterial Proteins↗
PubMed1989

Spectroscopic characterization of the nickel and iron-sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapsa roseopersicina. 1. Electron spin resonance spectroscopy.

The thermostable hydrogenase from Thiocapsa roseopersicina was examined by low-temperature ESR spectroscopy. Two types of signals were detected, from an oxidized iron-sulphur cluster and a nickel centre (Ni-A). In the oxidized protein additional signals were observed due to spin-spin interaction between the two paramagnetic centres. This interaction could be reversibly abolished by reduction to a redox potential below 105 mV. This implies that an additional redox centre is involved in the interaction, for which an Fe3+ ion is suggested. Reduction with hydrogen induced a second type of nickel ESR signal (Ni-C), corresponding to an intermediate redox state seen in other nickel hydrogenases. The Ni-C species was light-sensitive at cryogenic temperatures. At temperatures near to 4.2 K the Ni-C signal showed evidence of interaction with another paramagnetic centre, presumably a second iron-sulphur cluster. On reoxidation a signal due to a third Ni(III) species, Ni-B, increased in amplitude. These results establish that metal centres in the hydrogenase from T. roseopersicina are closely similar to those of the well-studied hydrogenase from Chromatium vinosum.

Chromatiaceae↗
PubMed1989

Spectroscopic characterization of the nickel and iron-sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapsa roseopersicina. 2. Electron spin-echo spectroscopy.

Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynthetic bacterium Thiocapsa roseopersicina, an enzyme which contains nickel and iron-sulphur clusters but no flavin. The linear electric field effect profile of the spectrum in the region of g = 2.01 indicated that the strong ESR signal in the oxidized protein is due to a [3Fe-4S] cluster. The electron spin-echo envelope of this spectrum was modulated by hyperfine interactions with 1H and 14N nuclei, probably from the polypeptide chain. The ESR spectrum of this species shows a complex pattern arising from spin-spin interaction with another paramagnet. When the protein was partially reduced by ascorbate plus phenazine methosulphate, the complexity of the spectrum was abolished but the form of the electron spin-echo envelope modulation (ESEEM) pattern was unchanged. This indicates that the reversible disappearance of the spin-spin interaction pattern on partial reduction is not due to cluster interconversion to a [4Fe-4S] cluster. In the ESR spectrum of nickel(III), weak hyperfine interactions with 1H and 14N were also observed by ESEEM. The nature of the interacting nuclei is discussed.

Chromatiaceae↗
PubMed1989

Immunological relationship among hydrogenases.

We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies. Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina. The results showed a strong relationship among the Ni-Fe dimeric hydrogenases. The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens. The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved. Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase. Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases. The two hydrogenases from C. pasteurianum were found to be quite different immunologically. The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed.

Antibodies↗
PubMed1979

On the physical origin of biological handedness.

In the racemic conglomerate crystallization of over 1,000 samples of D,L-sodium-ammonium tartrate the effect of 32P beta irradiation on the weight, optical activity, and crystallite size was measured. Both weight and optical activity showed a statistical dependence on the intensity of beta irradiation. The crystallite size is also affected by the presence of 32P. Asymmetric crystals are suggested to have been potential mediators between asymmetric parity violating forces and molecular asymmetry so that stereo-selective prebiotic chemical reactions involving crystals need not be considered 'chance' processes. No measurable difference in the energy content of optical isomers was found. An upper limit for the direct contribution of weak interactions to electromagnetic ones has been calculated. The mechanism of stereoselective crystal seeding by beta particles is discussed.

Crystallization↗