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Journal of Bacteriology, February 1999, p. 1256-1263, Vol. 181, No. 4
Institute of Molecular and Cellular
Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
Received 24 July 1998/Accepted 10 December 1998
The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase (EC 1.1.1.34) was purified 3,000-fold from
Streptomyces sp. strain CL190 to apparent homogeneity with
an overall yield of 2.1%. The purification procedure consisted of
(NH4)2SO4 precipitation, heat
treatment and anion exchange, hydrophobic interaction, and affinity
chromatographies. The molecular mass of the enzyme was estimated to be
41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
100 to 105 kDa by gel filtration chromatography, suggesting that the
enzyme is most likely to be a dimer. The enzyme showed a pH optimum of
around 7.2, with apparent Km values of 62 µM
for NADPH and 7.7 µM for HMG-CoA. A gene from CL190 responsible for
HMG-CoA reductase was cloned by the colony hybridization method with an
oligonucleotide probe synthesized on the basis of the N-terminal
sequence of the purified enzyme. The amino acid sequence of the CL190
HMG-CoA reductase revealed several limited motifs which were highly
conserved and common to the eucaryotic and archaebacterial enzymes.
These sequence conservations suggest a strong evolutionary pressure to
maintain amino acid residues at specific positions, indicating that the
conserved motifs might play important roles in the structural
conformation and/or catalytic properties of the enzyme.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Purification, Characterization, and Cloning of a
Eubacterial 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, a
Key Enzyme Involved in Biosynthesis of Terpenoids
*
Corresponding author. Mailing address: Institute of
Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku,
Tokyo 113-0032, Japan. Phone: 81-3-5684-2617. Fax: 81-3-3816-0453. E-mail: c00402{at}simail.ne.jp.
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