Purification and Characterization of NADH Dehydrogenase from Bacillus subtilis

Abstract

NADH dehydrogenase from Baicillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X‐100 by hydrophobic interaction chromatography on an octyl‐Sepharose CL‐4B column. A 70‐fold purification is achieved. No other components could be detected with sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin‐layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH: 3‐(4′‐5′‐dimethyl‐thiazol‐2‐yl)2,4‐diphenyltetrazolium bromide oxidoreduction is optimal at 37°C and pH 7.5–8.0. The purified enzyme has a Kapp for NADH of 60 μM and a V of 23.5 μmol NADH/min · mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH‐related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH.

13C-flux Analysis Reveals NADPH-balancing Transhydrogenation Cycles in Stationary Phase of Nitrogen-starving Bacillus subtilis

“…Although no genes were annotated for this reaction in B. subtilis, we found significant in vitro transhydrogenase activity in nitrogen starvation-induced resting wild-type that was only marginally lower in exponentially growing cells and similar in the ytsJ/gapB double mutant (Fig. 6A) (31,55,56). If this in vitro activity could be exploited in vivo, the transhydrogenase reaction could potentially contribute 1.1 Ϯ 0.1 mmol⅐g Ϫ1 ⅐h Ϫ1 or ϳ21% to NADPH balancing.…”

Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

“…Another enzyme, called malate:quinone oxidoreductase (MQO; EC 1.1.99.16), catalyzes the same reaction as MDH in Azotobacter (Jurtshuk et al, 1969), Mycobacterium (Yano et al, 2006), Micrococcus (Cohn, 1956), B. subtilis (Bergsma et al, 1982), C. glutamicum (Molenaar et al, 1998), E. coli , Pseudomonas aeruginosa (Kretzschmar et al, 2002), Pseudomonas citronellolis (Förster-Fromme and Jendrossek, 2005), and Helicobacter pylori (Kather et al, 2000). MQO is a membrane protein that catalyzes the oxidation of malate to oxaloacetate using NAD + as coenzyme and quinones as electron acceptors and the reaction is irreversible.…”

Electron Transfer Ability from NADH to Menaquinone and from NADPH to Oxygen of Type II NADH Dehydrogenase ofCorynebacterium glutamicum

“…These enzymes lack iron-sulfur clusters and contain a flavin, non-covalently bound FAD in most cases 13,15,16) but covalently bound FMN in other cases. 17,18) There is no evidence that NDH-2 from these microorganisms contains a metal binding motif.…”

“…8) Although bacterial NDH-1 from E. coli 9,10) and P. denitrificans 11,12) has been well characterized, NDH-2 has not been well studied, except for E. coli NDH-2. 13,14) Prokaryotic NDH-2 has been isolated from B. subtilis, 15) Methylococcus capsulatus, 16) Acidianus ambivalens, 17) and Sulfolobus metallicus.…”