Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus

Sang Suk Kim, In-Geol Choi, Sung Hou Kim, Yeon Gyu Yu

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts L- or D-glutamate to D- or L-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K(m) and k(cat) values of the overexpressed A. pyrophilus glutamate racemase for the racemization of L-glutamate to the D-form and the conversion of D-glutamate to the L-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06s-1 or 0.50 ± 0.07 mM and 0.25 ± 0.01 s-1, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min.

Original languageEnglish
Pages (from-to)175-183
Number of pages9
JournalExtremophiles
Volume3
Issue number3
DOIs
Publication statusPublished - 1999 Aug 1
Externally publishedYes

Fingerprint

glutamate racemase
Molecular Cloning
Racemases and Epimerases
Glutamic Acid
Bacteria
Cysteine
Lactobacillus brevis
Escherichia coli
L Forms
Amino Acids
Bacillus subtilis
Glutamine
Aspartic Acid
Alanine
Genes
Proteins
Salts
Phosphates
Ions

Keywords

  • Aquifex pyrophilus
  • Glutamate racemase
  • Molecular cloning
  • Overexpression
  • Thermostability

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Microbiology

Cite this

Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus. / Kim, Sang Suk; Choi, In-Geol; Kim, Sung Hou; Yu, Yeon Gyu.

In: Extremophiles, Vol. 3, No. 3, 01.08.1999, p. 175-183.

Research output: Contribution to journalArticle

@article{8fdc8ab819ad410583fef4f84982f77c,
title = "Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus",
abstract = "A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts L- or D-glutamate to D- or L-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K(m) and k(cat) values of the overexpressed A. pyrophilus glutamate racemase for the racemization of L-glutamate to the D-form and the conversion of D-glutamate to the L-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06s-1 or 0.50 ± 0.07 mM and 0.25 ± 0.01 s-1, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50{\%} of its activity after incubation at 85°C for 90 min.",
keywords = "Aquifex pyrophilus, Glutamate racemase, Molecular cloning, Overexpression, Thermostability",
author = "Kim, {Sang Suk} and In-Geol Choi and Kim, {Sung Hou} and Yu, {Yeon Gyu}",
year = "1999",
month = "8",
day = "1",
doi = "10.1007/s007920050114",
language = "English",
volume = "3",
pages = "175--183",
journal = "Extremophiles : life under extreme conditions",
issn = "1431-0651",
publisher = "Springer Japan",
number = "3",

}

TY - JOUR

T1 - Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus

AU - Kim, Sang Suk

AU - Choi, In-Geol

AU - Kim, Sung Hou

AU - Yu, Yeon Gyu

PY - 1999/8/1

Y1 - 1999/8/1

N2 - A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts L- or D-glutamate to D- or L-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K(m) and k(cat) values of the overexpressed A. pyrophilus glutamate racemase for the racemization of L-glutamate to the D-form and the conversion of D-glutamate to the L-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06s-1 or 0.50 ± 0.07 mM and 0.25 ± 0.01 s-1, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min.

AB - A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts L- or D-glutamate to D- or L-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K(m) and k(cat) values of the overexpressed A. pyrophilus glutamate racemase for the racemization of L-glutamate to the D-form and the conversion of D-glutamate to the L-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06s-1 or 0.50 ± 0.07 mM and 0.25 ± 0.01 s-1, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min.

KW - Aquifex pyrophilus

KW - Glutamate racemase

KW - Molecular cloning

KW - Overexpression

KW - Thermostability

UR - http://www.scopus.com/inward/record.url?scp=0000026654&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0000026654&partnerID=8YFLogxK

U2 - 10.1007/s007920050114

DO - 10.1007/s007920050114

M3 - Article

C2 - 10484173

AN - SCOPUS:0000026654

VL - 3

SP - 175

EP - 183

JO - Extremophiles : life under extreme conditions

JF - Extremophiles : life under extreme conditions

SN - 1431-0651

IS - 3

ER -