Further improvement of the thermal stability of a partially stabilized Bacillus subtilis 3-isopropylmalate dehydrogenase variant by random and site-directed mutagenesis

被引:43
作者
Akanuma, S
Yamagishi, A
Tanaka, N
Oshima, T
机构
[1] Tokyo Univ Pharm & Life Sci, Dept Mol Biol, Hachioji, Tokyo 1920392, Japan
[2] Tokyo Inst Technol, Dept Life Sci, Midori Ku, Yokohama, Kanagawa 227, Japan
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1999年 / 260卷 / 02期
关键词
3-isopropylmalate dehydrogenase; random mutagenesis; site-directed mutagenesis; thermal stability; Thermus thermophilus;
D O I
10.1046/j.1432-1327.1999.00182.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A thermostabilized mutant of Bacillus subtilis 3-isopropylmalate dehydrogenase (IPMDH) obtained in a previous study contained a set of triple amino acid substitutions. To further improve the stability of the mutant, we used a random mutagenesis technique and identified two additional thermostabilizing substitutions, Thr22--> Lys and Met256-->Val, that separately endowed the protein with further stability. We introduced the two mutations into a single enzyme molecule, thus constructing a mutant with overall quintuple mutations. Other studies have suggested that an improved hydrophobic subunit interaction and a rigid type II beta-turn play important roles in enhancing the protein stability. Based on those observations, we successively introduced amino acid substitutions into the mutant with the quintuple mutations by site-directed mutagenesis: Glu253 at the subunit interface was replaced by Leu to increase the hydrophobic interaction between the subunits; Glu112, Ser113 and Ser115 that were involved in the formation of the turn were replaced by Pro, Gly and Glu, respectively, to make the turn more rigid. The thermal stability of the mutants was determined based on remaining activity after heat treatment and first-order rate constant of thermal unfolding, which showed gradual increases in thermal stability as more mutations were included.
引用
收藏
页码:499 / 504
页数:6
相关论文
共 35 条
[1]   CONTROL OF OLIGOMERIC ENZYME THERMOSTABILITY BY PROTEIN ENGINEERING [J].
AHERN, TJ ;
CASAL, JI ;
PETSKO, GA ;
KLIBANOV, AM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1987, 84 (03) :675-679
[2]   Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution [J].
Akanuma, S ;
Yamagishi, A ;
Tanaka, N ;
Oshima, T .
PROTEIN SCIENCE, 1998, 7 (03) :698-705
[3]   HIGH-RESISTANCE OF ESCHERICHIA-COLI RIBONUCLEASE HI VARIANT WITH QUINTUPLE THERMOSTABILIZING MUTATIONS TO THERMAL-DENATURATION, ACID DENATURATION, AND PROTEOLYTIC DEGRADATION [J].
AKASAKO, A ;
HARUKI, M ;
OOBATAKE, M ;
KANAYA, S .
BIOCHEMISTRY, 1995, 34 (25) :8115-8122
[4]   SUBUNIT INTERFACE OF TRIOSEPHOSPHATE ISOMERASE - SITE-DIRECTED MUTAGENESIS AND CHARACTERIZATION OF THE ALTERED ENZYME [J].
CASAL, JI ;
AHERN, TJ ;
DAVENPORT, RC ;
PETSKO, GA ;
KLIBANOV, AM .
BIOCHEMISTRY, 1987, 26 (05) :1258-1264
[5]  
Chou P Y, 1978, Adv Enzymol Relat Areas Mol Biol, V47, P45
[6]   3-DIMENSIONAL STRUCTURE OF A HIGHLY THERMOSTABLE ENZYME, 3-ISOPROPYLMALATE DEHYDROGENASE OF THERMUS-THERMOPHILUS AT 2.2A RESOLUTION [J].
IMADA, K ;
SATO, M ;
TANAKA, N ;
KATSUBE, Y ;
MATSUURA, Y ;
OSHIMA, T .
JOURNAL OF MOLECULAR BIOLOGY, 1991, 222 (03) :725-738
[7]   THE NUCLEOTIDE-SEQUENCE OF 3-ISOPROPYLMALATE DEHYDROGENASE GENE FROM BACILLUS-SUBTILIS [J].
IMAI, R ;
SEKIGUCHI, T ;
NOSOH, Y ;
TSUDA, K .
NUCLEIC ACIDS RESEARCH, 1987, 15 (12) :4988-4988
[8]   LYSOZYME REQUIRES FLUCTUATION OF THE ACTIVE-SITE FOR THE MANIFESTATION OF ACTIVITY [J].
IMOTO, T ;
UEDA, T ;
TAMURA, T ;
ISAKARI, Y ;
ABE, Y ;
INOUE, M ;
MIKI, T ;
KAWANO, K ;
YAMADA, H .
PROTEIN ENGINEERING, 1994, 7 (06) :743-748
[9]  
KAGAWA Y, 1984, J BIOL CHEM, V259, P2956
[10]   SINGLE AMINO-ACID SUBSTITUTIONS CAN FURTHER INCREASE THE STABILITY OF A THERMOPHILIC L-LACTATE DEHYDROGENASE [J].
KALLWASS, HKW ;
SUREWICZ, WK ;
PARRIS, W ;
MACFARLANE, ELA ;
LUYTEN, MA ;
KAY, CM ;
GOLD, M ;
JONES, JB .
PROTEIN ENGINEERING, 1992, 5 (08) :769-774