Hydrogenases that are composed of two dissimilar subunits have been identified as beterodimeric (NiFe) or (NiFeSe) hydrogenases. Hydrogenases having more than two subunits are designated as multimeric (NiFe) or (NiFeSe) hydrogenases. The multimeric hydrogenases can be further subdivided on the basis of the involvement of the unique electron acceptors, F420 and NAD + as the F420-(NiFe) hydzogenases and the NAD+-(NiFe) hydrogenase. This connotation reflects the molecular relationships within the gene family and accommodates a number of biochemical realities. NICKEL HYDROOENASE FAMILY
Deletion mutants of Escherichia coli specific for hydrogenase isoenzyme 1 (HYD1) have been constructed and characterized. The hya operon, which contains genes for the two HYD1 structural subunits and four additional genes, was mapped at 22 min on the E. coli chromosome. The total hydrogenase activities of the HYD1-negative mutant and wild-type strains were similar. However, the formate dehydrogenase activity associated with the formate hydrogen lyase pathway was lower in the mutant. The hya mutant (strain AP1), complemented with only the hydrogenase structural genes (hyaAB), produced antigenically identifiable but inactive HYD1 protein.The first five genes of hya (hyaA to hyaE) were required for the synthesis of active HYD1, but wild-type levels of HYDl activity were restored only when mutant cells were transformed with all six genes of the operon. When AP1 was complemented with hya carried on a high-copy-number plasmid, the HYD1 structural subunits were overexpressed, but the excess protein was unprocessed and localized in the soluble fraction of the cell. The products of hyaDEF are postulated to be involved in the processing of nascent structural subunits (HYAA and HYAB). This processing takes place only after the subunits are inserted into the cell membrane. It is concluded that the biosynthesis of active HYD1 is a complex biochemical process involving the cellular localization and processing of nascent structural subunits, which are in turn dependent on the insertion of nickel into the nascent HYD1 large subunit.Hydrogen metabolism in Escherichia coli is tightly regulated by parameters of growth (2, 27) and involves three discrete nickel-containing hydrogenases: two electrophoretically stable, membrane-bound heterodimeric enzymes, hydrogenase 1 (HYD1) (28) and hydrogenase 2 (HYD2) (3), and a labile hydrogen-evolving hydrogenase, hydrogenase 3, which is as yet uncharacterized (27). HYD1 has been purified from anaerobically grown cells and biochemically characterized (28). It has a molecular mass of 200 kDa, is composed of two large (60-kDa) and two small (32-kDa) subunits, and contains 11 nonheme iron atoms and 1 g-atom of nickel per mol of enzyme (28 (16,24,25,30,33); and (iii) mutants unable to utilize hydrogen in the presence of electron acceptors (16,32). None of the mutants, however, have been shown to specifically impair HYD1 activity, indicating that the mutations are not in the hya operon. In this paper, we report the construction, biochemical characterization, and genetic analysis of HYD1-specific deletion mutants. MATERIALS AND METHODSBacterial strains and culture conditions. All bacterial strains used in this study are derivatives of E. coli K-12 and are listed in Table 1. Bacteria were cultured in Luria broth with 0.4% glucose as the carbon source (LBG). Antibiotics were added at final concentrations of 50 jig/ml (kanamycin), 100 jig/ml (ampicillin), and 20 ,ug/ml (chloramphenicol). For studying the incorporation of nickel into HYD1, cells were grown anaerobically in LBG in the presence of 1.2 ,iM...
DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.The anaerobic hydrogen metabolism of Escherichia coli and other enterobacteria is intricately regulated with regard to both hydrogen production during fermentation and hydrogen oxidation during anaerobic respiration (13,14). This complex regulatory system responds to specific substrates as well as to global regulatory signals and results in the biosynthesis of discrete hydrogenases specific to a given metabolic pathway. Three E. coli hydrogenases have been described which are synthesized in response to different physiological conditions. Hydrogenases 1 and 2 have been biochemically characterized and are immunologically distinct membranebound nickel-containing proteins (2, 3, 31). The existence of hydrogenase 3 was originally inferred from the fact that hydrogenases 1 and 2 immunoprecipitated from cell lysates did not account for total hydrogenase activity (30). Hydrogenase 3 activity is very labile and has been only partially characterized (34). Hydrogenases 1 and 3 are induced to higher levels by growth on glucose and formate. Hydrogenase 3 has also been shown to have a role in the formate hydrogenlyase pathway and accounts for about 60 to 70% of the total hydrogenase activity in the cell. The physiological role of hydrogenase 1 has not been defined. Hydrogenase 2 has been implicated as a respiratory uptake hydrogenase coupled to fumarate reduction and is induced to a higher level by growth in the presence of glycerol and fumarate (30).The presence of three different enzymes catalyzing the same reactions makes the biochemical, physiological, and genetic analyses of their metabolic roles technically difficult. Toward understanding the physiological roles of the different hydrogenases, a large number of E. coli mutants defective in hydrogenase activities have been analyzed. Most of these mutants lack all three hydrogenase activities; based on genetic analysis, the mutations appear to be exclusively at loci affecting regulation and do not encompass the hydrogenase structural genes. These genetic loci have been designated as hydA through F (8, 16...
Over the past decade, our understanding of cardiomyopathies has improved dramatically, due to improvements in screening and detection of gene defects in the human genome as well as a variety of novel animal models (mouse, zebrafish, and drosophila) and in silico computational models. These novel experimental tools have created a platform that is highly complementary to the naturally occurring cardiomyopathies in cats and dogs that had been available for some time. A fully integrative approach, which incorporates all these modalities, is likely required for significant steps forward in understanding the molecular underpinnings and pathogenesis of cardiomyopathies. Finally, novel technologies, including CRISPR/Cas9, which have already been proved to work in zebrafish, are currently being employed to engineer sarcomeric cardiomyopathy in larger animals, including pigs and non-human primates. In the mouse, the increased speed with which these techniques can be employed to engineer precise 'knock-in' models that previously took years to make via multiple rounds of homologous recombination-based gene targeting promises multiple and precise models of human cardiac disease for future study. Such novel genetically engineered animal models recapitulating human sarcomeric protein defects will help bridging the gap to translate therapeutic targets from small animal and in silico models to the human patient with sarcomeric cardiomyopathy.
Two electrophoreticforms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrro gigas have been detected by Western analysis The faster movmg form co-migrates with the large subunit from punfied, active enzyme. Amino acid sequence and composition of the C-terminal tryptic peptide of the large subumt from purified hydrogenase revealed that It is 15 amino acids shorter than that predIcted by the nucleotide sequence. Processing of the nascent large subunit occurs by C-terminal cleavage between H~s"~ and Val"'. residues which are highly conserved among [NiFe] hydrogenases.Mutagenesis of the analogous residues, Hls5*' and Val"', m the E co/i hydrogenase-1 (HYDI) large subunit resulted m significant decrease in processing and HYDl activity.Hydrogenase large subunit; C-Terminal processmg; Mutagenesis
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