Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

ABSTRACT The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100°C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.

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Menaquinone (Vitamin K2) Biosynthesis: Localization and Characterization of the menA Gene fromEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.180.10.2782-2787.1998 ◽

1998 ◽

Vol 180 (10) ◽

pp. 2782-2787 ◽

Cited By ~ 75

Author(s):

K. Suvarna ◽

D. Stevenson ◽

R. Meganathan ◽

M. E. S. Hudspeth

Keyword(s):

High Performance ◽

Promoter Sequence ◽

Anaerobic Growth ◽

Gene Encoding ◽

Reading Frame ◽

Chromatography Analysis ◽

Naphthoic Acid ◽

Membrane Bound ◽

Carbon Side Chain

ABSTRACT A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of amenA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from theorf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into theorf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.

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Characterization of a gene encoding alcohol dehydrogenase in benznidazole-susceptible and -resistant populations of Trypanosoma cruzi

Acta Tropica ◽

10.1016/j.actatropica.2009.02.007 ◽

2009 ◽

Vol 111 (1) ◽

pp. 56-63 ◽

Cited By ~ 11

Author(s):

Fernanda M.F. Campos ◽

Daniel B. Liarte ◽

Renato A. Mortara ◽

Alvaro J. Romanha ◽

Silvane M.F. Murta

Keyword(s):

Alcohol Dehydrogenase ◽

Trypanosoma Cruzi ◽

Gene Encoding

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Characterization of Energy-Conserving Hydrogenase B in Methanococcus maripaludis

Journal of Bacteriology ◽

10.1128/jb.01446-09 ◽

2010 ◽

Vol 192 (15) ◽

pp. 4022-4030 ◽

Cited By ~ 27

Author(s):

Tiffany A. Major ◽

Yuchen Liu ◽

William B. Whitman

Keyword(s):

Gene Replacement ◽

Methanococcus Maripaludis ◽

Gene Encoding ◽

Western Blots ◽

Growth Defects ◽

Energy Conserving ◽

Membrane Bound ◽

Membrane Spanning ◽

And Function

ABSTRACT The Methanococcus maripaludis energy-conserving hydrogenase B (Ehb) generates low potential electrons required for autotrophic CO2 assimilation. To analyze the importance of individual subunits in Ehb structure and function, markerless in-frame deletions were constructed in a number of M. maripaludis ehb genes. These genes encode the large and small hydrogenase subunits (ehbN and ehbM, respectively), a polyferredoxin and ferredoxin (ehbK and ehbL, respectively), and an ion translocator (ehbF). In addition, a gene replacement mutation was constructed for a gene encoding a putative membrane-spanning subunit (ehbO). When grown in minimal medium plus acetate (McA), all ehb mutants had severe growth deficiencies except the ΔehbO::pac strain. The membrane-spanning ion translocator (ΔehbF) and the large hydrogenase subunit (ΔehbN) deletion strains displayed the severest growth defects. Deletion of the ehbN gene was of particular interest because this gene was not contiguous to the ehb operon. In-gel activity assays and Western blots confirmed that EhbN was part of the membrane-bound Ehb hydrogenase complex. The ΔehbN strain was also sensitive to growth inhibition by aryl acids, indicating that Ehb was coupled to the indolepyruvate oxidoreductase (Ior), further supporting the hypothesis that Ehb provides low potential reductants for the anabolic oxidoreductases in M. maripaludis.

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