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Archives of Microbiology

, Volume 154, Issue 6, pp 605–613 | Cite as

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

  • Matthias Nagel
  • Jan R. Andreesen
Original Papers

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.

Key words

Bacillus niacini Bactopterin 6-Hydroxynicotinate dehydrogenase Molybdenum Nicotinate dehydrogenase 

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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Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Matthias Nagel
    • 1
  • Jan R. Andreesen
    • 1
  1. 1.Institut für Mikrobiologic der UniversitätGöttingenFederal Republic of Germany

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