NADH dehydrogenase of Corynebacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH

Abstract

NADPH oxidase activity, in addition to NADH oxidase activity, has been shown to be present in the respiratory chain of Corynebacterium glutamicum. In this study, we tried to purify NADPH oxidase and NADH dehydrogenase activities from the membranes of C. glutamicum. Both the enzyme activities were simultaneously purified in the same fraction, and the purified enzyme was shown to be a single polypeptide of 55 kDa. The N-terminal sequence of the enzyme was consistent with the sequence deduced from the NADH dehydrogenase gene of C. glutamicum, which has been sequenced and shown to be a homolog of NADH dehydrogenase II. In addition to high NADH-ubiquinone-1 oxidoreductase activity at neutral pH, the purified enzyme showed relatively high NADPH oxidase and NADPH-ubiquinone-1 oxidoreductase activities at acidic pH. Thus, NADH dehydrogenase of C. glutamicum was shown to be rather unique in having a relatively high reactivity toward NADPH.

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

“…In addition, D213N and D213Q variants of C. glutamicum NDH-II were constructed, and crude extracts of C. glutamicum ⌬ndh (carrying pVWEx1-ndh D213N or pVWEx1-ndh D213Q ) were prepared and analyzed. As reported previously (53), the crude extracts of the wild-type strain had no detectable ubiquinone oxidoreductase activity with NADPH but did show activity with NADH (k cat , 88 s Ϫ1 ; K m , 50 M) ( (Table 5). Taking the findings together, neutral amino acid residues (G, N, or Q) at position 213 of C. glutamicum NDH-II resulted in activities with NADPH that were comparable to, or higher than, those than with NADH.…”

“…NDH-II has two conserved flavin adenine dinucleotide (FAD) binding domains with a glycine-rich consensus sequence (GXGXXG) along with an NADH binding domain containing a negatively charged residue (D or E) at the end of the second ␤-sheet, which determines the nucleotide specificity (55). Previous studies revealed that NDH-II was solely responsible for NADH oxidation (NADH:ubiquinone oxidoreductase), with maximal activity at pH 6.5, whereas NADPH:ubiquinone oxidoreductase activity was detectable in vitro at pH 4.5 but negligible at pH 6.5 (53).…”

Comparative analysis of the accelerated aged seed transcriptome profiles of two maize chromosome segment substitution lines

“…DEG14 encodes an HSP20-like chaperone superfamily protein. DEG15 encodes an NADH dehydrogenase subunit 4, an important enzyme in the electron-transport chain in mitochondria [47] . DEG16 encodes an embryo defective 3012.…”