Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from Corynebacterium glutamicum

被引:15
作者
Hsu, SK
Lin, LL
Lo, HH
Hsu, WH [1 ]
机构
[1] Natl Chung Hsing Univ, Inst Mol Biol, Taichung 402, Taiwan
[2] Chungtai Inst Hlth Sci & Technol, Dept Med Technol, Taichung 406, Taiwan
[3] Natl Chiayi Univ, Dept Appl Chem, Chiayi 60083, Taiwan
[4] Chungtai Inst Hlth Sci & Technol, Dept Dent Technol, Taichung 406, Taiwan
关键词
prephenate dehydratase; site-directed mutagenesis; feedback inhibition; catalytic activity; Corynebacterium glutamicum;
D O I
10.1007/s00203-004-0649-5
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 muM phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K-m and k(cat) values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K-m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K-m and k(cat) values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.
引用
收藏
页码:237 / 244
页数:8
相关论文
共 41 条
[11]  
DOPHEIDE TA, 1972, J BIOL CHEM, V247, P4447
[12]   Cation-pi interactions in chemistry and biology: A new view of benzene, Phe, Tyr, and Trp [J].
Dougherty, DA .
SCIENCE, 1996, 271 (5246) :163-168
[13]   PREPHENATE DEHYDRATASE OF THE ACTINOMYCETE AMYCOLATOPSIS-METHANOLICA - PURIFICATION AND CHARACTERIZATION OF WILD-TYPE AND DEREGULATED MUTANT PROTEINS [J].
EUVERINK, GJW ;
WOLTERS, DJ ;
DIJKHUIZEN, L .
BIOCHEMICAL JOURNAL, 1995, 308 :313-320
[14]   REGULATION OF PREPHENATE DEHYDRATASE IN CORYNEFORM SPECIES OF BACTERIA BY L-PHENYLALANINE AND BY REMOTE EFFECTORS [J].
FAZEL, AM ;
JENSEN, RA .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1980, 200 (01) :165-176
[15]   Energetics of a hydrogen bond (charged and neutral) and of a cation-π interaction in apoflavodoxin [J].
Fernández-Recio, J ;
Romero, A ;
Sancho, J .
JOURNAL OF MOLECULAR BIOLOGY, 1999, 290 (01) :319-330
[16]   MOLECULAR-CLONING AND NUCLEOTIDE-SEQUENCE OF THE CORYNEBACTERIUM-GLUTAMICUM-PHEA GENE [J].
FOLLETTIE, MT ;
SINSKEY, AJ .
JOURNAL OF BACTERIOLOGY, 1986, 167 (02) :695-702
[17]   CHORISMATE MUTASE-PREPHENATE DEHYDRATASE FROM ESCHERICHIA-COLI-K12 .2. EVIDENCE FOR IDENTICAL SUBUNITS CATALYZING 2 ACTIVITIES [J].
GETHING, MJH ;
DAVIDSON, BE .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1976, 71 (02) :327-336
[18]   PRODUCTION OF AROMATIC AMINO-ACIDS BY MICROORGANISMS .4. L-PHENYLALANINE PRODUCTION BY ANALOG-RESISTANT MUTANTS OF CORYNEBACTERIUM-GLUTAMICUM [J].
HAGINO, H ;
NAKAYAMA, K .
AGRICULTURAL AND BIOLOGICAL CHEMISTRY, 1974, 38 (01) :157-161
[19]   METABOLIC ENGINEERING TO PRODUCE TYROSINE OR PHENYLALANINE IN A TRYPTOPHAN-PRODUCING CORYNEBACTERIUM-GLUTAMICUM STRAIN [J].
IKEDA, M ;
KATSUMATA, R .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1992, 58 (03) :781-785
[20]   RECENT ADVANCES IN THE PHYSIOLOGY AND GENETICS OF AMINO ACID-PRODUCING BACTERIA [J].
JETTEN, MSM ;
SINSKEY, AJ .
CRITICAL REVIEWS IN BIOTECHNOLOGY, 1995, 15 (01) :73-103