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Purification and characterization of the molybdoenzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini

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Abstract

The enzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini could be purified to homogeneity by means of anion exchange chromatography, hydrophobic interaction chromatography, gel filtration, and chromatography on hydroxylapatite. During enrichment procedures both enzymes showed a significant loss in specific activity. The molecular weight of nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase was determined to be about 300,000 and 120,000, respectively. They were highly substrate specific and transferred electrons only to artificial acceptors of high redox potential. The K m for their specific substrates was about 1.0 mM for both enzymes, and their pH optimum was determined to be 7.5. For nicotinate dehydrogenase a content of 8.3 mol iron, 1.5 mol acid-labile sulfur, 2.0 mol flavin, and 1.5 mol molybdenum per mol of enzyme was determined. Both enzymes contained FAD and Fe/S center. After inhibition by KCN, thiocyanate was detected, and subsequently the initial nicotinate dehydrogenase activity was restored by the addition of Na2S indicating the presence of cyanolyzable sulfur. 6-Hydroxynicotinate dehydrogenase seemed to contain the same type of constituents as determined for nicotinate dehydrogenase. A partial immunological identity of the enzymes could be shown by antibodies raised against nicotinate dehydrogenase.

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Abbreviations

DCPIP:

2,6-dichlorophenol-indophenol

EEO:

electroendosmosis

FTTC:

fluorescein isothiocyanate

HAP:

hydroxylapatite

6-HDH:

6-hydroxynicotinate dehydrogenase

NBT:

nitroblue tetrazolium chloride

NDH:

nicotinate dehydrogenase

MTT:

thiazolyl blue

PES:

phenazine ethosulfate

PMSF:

phenylmethyl sulfonyl fluoride

TEMED:

N,N,N',N'-tetramethyl-ethylenediamine

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  1. Institut für Mikrobiologic der Universität, Grisebachstrasse 8, W-3400, Göttingen, Federal Republic of Germany

    Matthias Nagel & Jan R. Andreesen

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  1. Matthias Nagel
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Nagel, M., Andreesen, J.R. Purification and characterization of the molybdoenzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini . Arch. Microbiol. 154, 605–613 (1990). https://doi.org/10.1007/BF00248844

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