Purification and characterization of the oxygen-sensitive acetohydroxy acid synthase from the archaebacterium Methanococcus aeolicus.
Acetohydroxy acid synthase (EC 4.1.3.18) of the archaebacterium Methanococcus aeolicus was purified 1,150-fold to homogeneity. The molecular weight of the purified enzyme was 125,000, and it contained only one type of subunit (M(r) = 58,000). The amino-terminal sequence had 46 to 57% similarity to those of the large subunits of the eubacterial anabolic enzymes and 37 to 43% similarity to those of the yeast and plant enzymes. The methanococcal enzyme had a pH optimum of 7.6. The pI, estimated by chromatofocusing, was 5.6. Activity required Mg2+ or Mn2+ ions, thiamine pyrophosphate, and a flavin. Flavin adenine dinucleotide, flavin mononucleotide, and riboflavin plus 10 mM phosphate all supported activity. However, activity was strongly inhibited by these flavins at 0.3 mM. The Michaelis constants for pyruvate, MgCl2, MnCl2, thiamine pyrophosphate, flavin adenine dinucleotide, and flavin mononucleotide were 6.8 mM, 0.3 mM, 0.16 mM, 1.6 microM, 0.4 microM, and 1.3 microM, respectively. In cell extracts, the enzyme was sensitive to O2 (half-life = 2.7 min with 5% O2 in the headspace), but the purified enzyme was less sensitive to O2 (half-life = 78.0 min with 20% O2). Reconstitution of the enzyme with flavin adenine dinucleotide increased the sensitivity to O2. Moreover, in the assay the homogeneous enzyme was rapidly inactivated by O2, and the concentration required for 50% inhibition (I50) was obtained with an atmosphere of 0.11% O2. The methanococcal enzyme has similarities to the eubacterial and eucaryotic enzymes, consistent with the ancient origin of the archaebacterial enzyme.
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Department of Microbiology, University of Georgia, Athens 30602-2605.
Whitman
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D000095:Acetolactate Synthase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000378:metabolism / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D017017:Methanococcus / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009994:Osmolar Concentration; D010100:Oxygen / Q000633:toxicity*; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation
D005415:Flavins; D008345:Manganese; D017886:Durapatite; D000095:Acetolactate Synthase; D008274:Magnesium; D010100:Oxygen