The organization of hydrogenase in the cytoplasmic membrane of <i>Escherichia coli</i>

Graham, Alexander

doi:10.1042/bj1970283

Cited by 16 publications

(15 citation statements)

References 21 publications

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“…The core components of each enzyme are a large subunit that contains the Ni–Fe active site and a small subunit which binds iron–sulfur clusters. The active sites of hydrogenases 1 and 2 are at the periplasmic face of the membrane, where they catalyse respiratory hydrogen oxidation (Graham, 1981; Rodrigue et al ., 1996; Sargent et al ., 1998). The hydrogenase 1 and 2 small subunit precursors have twin arginine signal peptides (Menon et al ., 1990; Sargent et al ., 1998), while the large subunits, despite their periplasmic location, lack export signals.…”

Section: Resultsmentioning

confidence: 99%

Overlapping functions of components of a bacterial Sec-independent protein export pathway

et al. 1998

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We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.

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Section: Resultsmentioning

confidence: 99%

Overlapping functions of components of a bacterial Sec-independent protein export pathway

et al. 1998

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“…Boxer and co-workers (4, 28) purified HYD1 and an active fragment of HYD2 and reported that these two isoenzymes are immunologically distinct. Other descriptions of hydrogenase purification from E. coli, to different levels of purity, can be found in the literature (1,5,13,14,31). Hydrogenases that had been purified to homogeneity had apparent molecular weights of 56,000 (1), 58,000 (14), and 200,000 (28).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 1990

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A hydrogenase associated with dihydrogen uptake (HUP hydrogenase) was purified from an Escherichia coli mutant (strain SE1100) defective in utilization of molybdate and thus fermentative dihydrogen production. This protein had two subunits with apparent molecular weights of 59,000 and 28,000 (form 1). An immunologically cross-reactive hydrogenase was also purified from E. coli K10 grown in glucose-minimal medium and harvested at the mid-exponential phase of growth. Upon purification to homogeneity, this hydrogenase contained only one subunit with an apparent molecular weight of 59,000 (form 2). The two forms of the HUP hydrogenase exhibited similar kinetic characteristics. The electrophoretic properties of the enzyme and its response to pH suggest that this HUP hydrogenase is the HYD1 isoenzyme. The HYD1 isoenzyme was the only hydrogenase detectable during the stationary phase of growth in E. coli grown in Mo-deficient medium.

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“…Furthermore, their structures within the membrane must be radically different for although the 35-kDa polypeptide of each is susceptible to tryptic cleavage in their membrane-bound forms, this treatment gives rise to a soluble active fragment only in the case of isoenzyme 2. Previous work from this laboratory has shown that the 64-kDa polypeptide of isoenzyme 1 is transmembraneously situated in the membrane [3,24]. The release of the tryptic derivative of isoenzyme 2 from the membrane indicates that its 61-kDa subunit and the majority of its 35-kDa subunit must be situated in the membrane in a position which is external to the membrane's permeability barrier.…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli

1986

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An active tryptic fragment of membrane‐bound hydrogenase isoenzyme 2 from anaerobically grown Escherichia coli has been purified. The soluble enzyme derivative was released from the membrane fraction by trypsin cleavage. The purification procedure involved ion‐exchange, hydroxyapatite and gel permeation chromatography. The enzyme derivative was purified 100‐fold from the membrane fraction and the specific activity of the final preparation was 320 μmol benzyl viologen reduced min−1 mg protein−1 (H2: benzyl viologen oxidoreductase). The native enzyme derivative had an Mr of 180000 and was composed of equimolar amounts of polypeptides of Mr 61000 and 30000. It possessed 12.5 mol Fe, 12.8 mol acid‐labile S2− and 3.1 mol Ni/180000 g enzyme. Antibodies were raised to the purified preparation which cross‐reacted with hydrogenase isoenzyme 2 but not with isoenzyme 1 in detergent‐dispersed preparations. Western immunoblot analysis revealed that isoenzyme 2 which had not been exposed to trypsin contained cross‐reacting polypeptides of Mr 61000 and 35000. Trypsin treatment of the membrane‐bound enzyme to form the soluble derivative of isoenzyme 2, therefore, cleaves a polypeptide of Mr 35000 to produce the 30000‐Mr fragment. Trypsin treatment of the detergent‐dispersed isoenzyme 2 produces the same fragmentation of the enzyme. Neither of the subunits of the enzyme revealed any immunological identity with those of hydrogenase isoenzyme 1.

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The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli

Cited by 16 publications

References 21 publications

Overlapping functions of components of a bacterial Sec-independent protein export pathway

Overlapping functions of components of a bacterial Sec-independent protein export pathway

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

Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli

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