Purification and characterization of two forms of hydrogenase isoenzyme 1 from Escherichia coli

Francis, Kevin; Patel, P. S.; Wendt, Jennifer; Shanmugam, K. T.

doi:10.1128/jb.172.10.5750-5757.1990

Cited by 31 publications

(30 citation statements)

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“…Comparison of the nucleotide-derived amino acid sequence of the small subunit of HYD1 with the NH2-terminal amino acid sequence of the mature small subunit, as determined by protein sequencing (8), indicates the presence of a long mitochondrionlike signal peptide (22). The large subunit lacks a recognizable NH2-terminal signal peptide, since the NH2-terminal protein sequence (8) and the nucleotide-derived protein sequence are colinear (22).…”

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Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1

et al. 1991

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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...

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Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1

et al. 1991

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“…It has been shown in Escherichia coli that the processing of the small subunit involves the removal of the N-terminal signal peptide (7). Processing of the hydrogenase large subunit has been studied in Azotobacter vinelandii (13) and Desulfovibrio gigas (31).…”

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Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits

et al. 1994

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Rhizobium leguminosarum bv. viciae UPM791 induces the synthesis of an [NiFe] hydrogenase in pea (Pisum sativum L.) bacteroids which oxidizes the H2 generated by the nitrogenase complex-inside the root nodules. The synthesis of this hydrogenase requires the genes for the small and large hydrogenase subunits (hupS and hupL, respectively) and 15 accessory genes clustered in a complex locus in the symbiotic plasmid. We show here that the bacteroid hydrogenase activity is limited by the availability of nickel to pea plants. Addition of Ni2+ to plant nutrient solutions (up to 10 mg/liter) resulted in sharp increases (up to 15-fold) in hydrogenase activity. This effect was not detected when other divalent cations (Zn2+, Co2+, Fe2+, and Mn2+) were added at the same concentrations. Determinations of the steady-state levels of hupSL-specific mRNA indicated that this increase in hydrogenase activity was not due to stimulation of transcription of structural genes. Immunoblot analysis with antibodies raised against the large and small subunits of the hydrogenase enzyme demonstrated that in the low-nickel situation, both subunits are mainly present in slow-migrating, unprocessed forms. Supplementation of the plant nutrient solution with increasing nickel concentrations caused the conversion of the slow-migrating forms of both subunits into fast-moving, mature forms. This nickel-dependent maturation process of the hydrogenase subunits is mediated by accessory gene products, since bacteroids from H2 uptake-deficient mutants carrying TnS insertions in hupG and hupK and in hypB and hypE accumulated the immature forms of both hydrogenase subunits even in the presence of high nickel levels.Most hydrogen uptake hydrogenases are membrane-bound, heterodimeric iron-sulfur proteins containing nickel ([NiFe] hydrogenases) (for reviews, see references 9, 37, and 46). Bacteria forming nodules on legume roots synthesize uptake hydrogenases which recycle the H2 evolved by nitrogenase in the nodules (5, 27) and contribute to the overall efficiency of the N2 fixation process (6). These bacteria include Rhizobium leguminosarum bv. viciae and Bradyrhizobium japonicum, the microsymbionts of peas and soybeans, respectively. The genetic determinants for H2 uptake (hup genes) in R. leguminosarum bv. viciae UPM791 are clustered in a 20-kb DNA region of the symbiotic plasmid and have been isolated in cosmid pAL618 (24). This cosmid has the capacity to confer H2 uptake activity on Hup-strains of R. leguminosarum bv. viciae and Rhizobium etli in symbiosis with peas and beans, respectively (25). The DNA region spanning the H2 uptake gene cluster has been sequenced, and 17 genes, closely linked and oriented in the same direction, were identified (15,38,40). The first two genes, hupS and hupL, encode the hydrogenase structural polypeptides (14). The predicted proteins for the small (360 amino acid residues, including a leader peptide of 45 residues) and the large (596 amino acid residues) subunits are homologous to the corresponding hydrogenase structur...

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“…HYDl has also been purified and shown to consist of a large (60 kDa) subunit and a small (30 kDa) subunit (16,40). An active form of HYD1 containing only the large subunit has also been purified and characterized (1,16). The operon encoding the two structural subunits of HYD1 (hya) contains a total of six genes and has been mapped at 22 min on the E. coli chromosome (30,31).…”

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“…Although mutants defective in H2 uptake have been described (23,25), detailed analysis of the operon encoding HYD2 has not been carried out. HYDl has also been purified and shown to consist of a large (60 kDa) subunit and a small (30 kDa) subunit (16,40). An active form of HYD1 containing only the large subunit has also been purified and characterized (1, 16).…”

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Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2

et al. 1994

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The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced. They occur in an operon (hyb) which contains seven open reading frames. An hyb deletion mutant (strain AP3) failed to grow on dihydrogen-fumarate medium and also produced very low levels of HIYD1. All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3.The hyb operon was mapped at 65 min on the E. coli chromosome.Under anaerobic growth conditions, Escherichia coli produces three different nickel-containing hydrogenases (3, 39). Hydrogenase 3 (HYD3) is part of the formate hydrogenlyase (FHL) complex and is responsible for formate-dependent dihydrogen (H2) evolution. The operon encoding HYD3 and other accessory electron transport components of the FHL complex, hyc, has been identified and is located at 58 min on the E. coli chromosome (7). The highly oxygen-labile nature of HYD3 has precluded detailed biochemical characterization. HYD2 is involved in H2 uptake and can be differentially induced to high levels when cells are grown in medium containing H2 as an electron donor and fumarate as an electron acceptor (3,23,25,39). An active component of HYD2 has been purified and shown to be a heterodimeric enzyme with a 58-kDa large subunit and a 30-kDa small subunit (4). Although mutants defective in H2 uptake have been described (23,25), detailed analysis of the operon encoding HYD2 has not been carried out. HYDl has also been purified and shown to consist of a large (60 kDa) subunit and a small (30 kDa) subunit (16,40). An active form of HYD1 containing only the large subunit has also been purified and characterized (1, 16). The operon encoding the two structural subunits of HYD1 (hya) contains a total of six genes and has been mapped at 22 min on the E. coli chromosome (30,31). The function of HYD1 is not understood, but it is believed to have a role in hydrogen cycling during fermentative growth. In addition to the operons coding for the structural components of the three hydrogenases, a fourth operon, hyp, located at 58 min, is essential for activity of all three hydrogenases (20,26,38). At least one of the genes in this operon (hypB) is involved in nickel metabolism, most probably via nickel insertion into apoenzyme (27).In this paper, we present the DNA sequence of the operon encoding HYD2 (hyb), which contains seven open reading frames (ORFs). Cassette mutagenesis of the hyb operon on the chromosome resulted in a total loss of HYD2 expression and activity, as well as in significant reduction in HYD1 activity. MATERIALS AND METHODSBacterial strains. All bacterial strains used were E. coli K-12 derivatives and are listed in Table 1 [pH 7.0]), resuspended in the same buffer to an optical density of 0.5 at 600 nm, and used for whole-cell enzyme assays. Cell extracts were prepared by sonicating cell suspensions on ice with a model W385 sonicator (Heat Systems) for 20 5-s bursts. Triton X-100 was added to a final concentration of 2% (vol/vol), when req...

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Purification and characterization of two forms of hydrogenase isoenzyme 1 from Escherichia coli

Cited by 31 publications

References 34 publications

Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1

Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1

Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits

Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2

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