Previous Article | Next Article ![]()
Journal of Bacteriology, February 2000, p. 891-897, Vol. 182, No. 4
Institute of Molecular and Cellular
Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
Received 31 August 1999/Accepted 18 November 1999
In addition to the ubiquitous mevalonate pathway,
Streptomyces sp. strain CL190 utilizes the nonmevalonate
pathway for isopentenyl diphosphate biosynthesis. The initial step of
this nonmevalonate pathway is the formation of
1-deoxy-D-xylulose 5-phosphate (DXP) by condensation of
pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase. The
corresponding gene, dxs, was cloned from CL190 by using PCR
with two oligonucleotide primers synthesized on the basis of two highly
conserved regions among dxs homologs from six genera. The
dxs gene of CL190 encodes 631 amino acid residues with a
predicted molecular mass of 68 kDa. The recombinant enzyme
overexpressed in Escherichia coli was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and 130 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of 9.0, with a Vmax of 370 U per mg of protein and Kms of 65 µM for
pyruvate and 120 µM for D-glyceraldehyde 3-phosphate. The
purified enzyme catalyzed the formation of 1-deoxyxylulose by
condensation of pyruvate and glyceraldehyde as well, with a Km value of 35 mM for
D-glyceraldehyde. To compare the enzymatic properties of
CL190 and E. coli DXP synthases, the latter enzyme was also
overexpressed and purified. Although these two enzymes had different
origins, they showed the same enzymatic properties.
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cloning and Characterization of
1-Deoxy-D-Xylulose 5-Phosphate Synthase from
Streptomyces sp. Strain CL190, Which Uses both the
Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate
Biosynthesis
and
*
Corresponding author. Mailing address: Institute of
Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku,
Tokyo 113-0032, Japan. Phone: 81-3-5841-7839. Fax: 81-3-5841-8485. E-mail: haseto{at}imcbns.iam.u-tokyo.ac.jp.
Present address: Department of Biochemistry, Chiba University,
School of Medicine, Inohana, Chiba 260-8670, Japan.
This article has been cited by other articles:
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |