Properties of an NAD(H)-containing methanol dehydrogenase and its activator protein from Bacillus methanolicus.

Oxidation of C1-C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43,000-Mr subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Zn2+ and 1-2 Mg2+ ions. The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme.

Control of glycolytic flux in Zymomonas mobilis by glucose 6-phosphate dehydrogenase activity.

Glycolytic genes in Zymomonas mobilis are highly expressed and constitute half of the cytoplasmic protein. The first four genes (glf, zwf, edd, glk) in this pathway form an operon encoding a glucose permease, glucose 6-phosphate dehydrogenase (G6-P dehydrogenase), 6-phosphogluconate dehydratase, and glucokinase, respectively. Each gene was overexpressed from a tac promoter to investigate the control of glycolysis during the early stages of batch fermentation when flux (qCO(2)) is highest.

Reconstruction of glucose uptake and phosphorylation in a glucose-negative mutant of Escherichia coli by using Zymomonas mobilis genes encoding the glucose facilitator protein and glucokinase.

Expression of the Zymomonas mobilis glf (glucose facilitator protein) and glk (glucokinase) genes in Escherichia coli ZSC113 (glucose negative) provided a new functional pathway for glucose uptake and phosphorylation. Both genes were essential for the restoration of growth in glucose minimal medium and for acid production on glucose-MacConkey agar plates.

Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19.

The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.

Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentative genes in Escherichia coli and Zymomonas mobilis.

The Zymomonas mobilis genes encoding alcohol dehydrogenase I (adhA), alcohol dehydrogenase II (adhB), and pyruvate decarboxylase (pdc) were overexpressed in Escherichia coli and Z. mobilis by using a broad-host-range vector containing the tac promoter and the lacIq repressor gene. Maximal IPTG (isopropyl-beta-D-thiogalactopyranoside) induction of these plasmid-borne genes in Z.