Reaction kinetic pathway of the recombinant octaprenyl pyrophosphate synthase from Thermotoga maritima:: how is it different from that of the mesophilic enzyme

被引:9
作者
Kuo, TH
Liang, PH
机构
[1] Acad Sinica, Inst Biol Chem, Taipei 11529, Taiwan
[2] Natl Taiwan Univ, Inst Biochem Sci, Taipei 10098, Taiwan
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS | 2002年 / 1599卷 / 1-2期
关键词
prenyltransferase; ubiquinone; single-turnover; thermophile; mesophile;
D O I
10.1016/S1570-9639(02)00410-7
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
Octaprenyl pyrophosphate synthase (OPPs) catalyzes the chain elongation of farnesyl pyrophosphate (FPP) via consecutive condensation reactions with five molecules of isopentenyl pyrophosphate (IPP) to generate all-trans C-40-octaprenyl pyrophosphate. The polymer forms the side chain of ubiquinone that is involved in electron transport system to produce ATP. Our previous study has demonstrated that Escherichia coli OPPs catalyzes IPP condensation with a rate of 2 s(-1) but product release limits the steady-state rate at 0.02 s(-1) [Biochim. Biophys. Acta 1594 (2002) 64]. In the present studies, a putative gene encoding for OPPs from Thermotoga maritima, an anaerobic and thermophilic bacterium, was expressed, purified, and its kinetic pathway was determined. The enzyme activity at 25 degreesC was 0.005 s(-1) under steady-state condition and was exponentially increased with elevated temperature. In contrast to E. coli OPPs, IPP condensation rather than product release was rate limiting in enzyme reaction. The product of chain elongation catalyzed by T maritima OPPs was C-40 and the rate of its conversion to C-45 was negligible. Under single-turnover condition with 10 muM OPPs(.)FPP complex and 1 muM IPP, only the C-20 was formed. rather than C-20-C-40 observed for E. coli enzyme. Together, our data suggest that the thermophilic OPPs from T maritima has lower enzyme activity at 25 degreesC, higher product specificity, higher thermal stability and lower structural flexibility than its mesophilic counterpart from E. coli. (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:125 / 133
页数:9
相关论文
共 28 条
[1]   ENZYMES AND PROTEINS FROM ORGANISMS THAT GROW NEAR AND ABOVE 100-DEGREES-C [J].
ADAMS, MWW .
ANNUAL REVIEW OF MICROBIOLOGY, 1993, 47 :627-658
[2]   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J].
Altschul, SF ;
Madden, TL ;
Schaffer, AA ;
Zhang, JH ;
Zhang, Z ;
Miller, W ;
Lipman, DJ .
NUCLEIC ACIDS RESEARCH, 1997, 25 (17) :3389-3402
[3]   THE IDENTIFICATION OF ESCHERICHIA-COLI ISPB (CEL) GENE ENCODING THE OCTAPRENYL DIPHOSPHATE SYNTHASE [J].
ASAI, K ;
FUJISAKI, S ;
NISHIMURA, Y ;
NISHINO, T ;
OKADA, K ;
NAKAGAWA, T ;
KAWAMUKAI, M ;
MATSUDA, H .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1994, 202 (01) :340-345
[4]  
ASHBY MN, 1990, J BIOL CHEM, V265, P13157
[5]   ANALYSIS OF NUMERICAL-METHODS FOR COMPUTER-SIMULATION OF KINETIC PROCESSES - DEVELOPMENT OF KINSIM - A FLEXIBLE, PORTABLE SYSTEM [J].
BARSHOP, BA ;
WRENN, RF ;
FRIEDEN, C .
ANALYTICAL BIOCHEMISTRY, 1983, 130 (01) :134-145
[6]   Probing the conformational change of Escherichia coli undecaprenyl pyrophosphate synthase during catalysis using an inhibitor and tryptophan mutants [J].
Chen, YH ;
Chen, APC ;
Chen, CT ;
Wang, AHJ ;
Liang, PH .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (09) :7369-7376
[7]   DISTRIBUTION OF ISOPRENOID QUINONE STRUCTURAL TYPES IN BACTERIA AND THEIR TAXONOMIC IMPLICATIONS [J].
COLLINS, MD ;
JONES, D .
MICROBIOLOGICAL REVIEWS, 1981, 45 (02) :316-354
[8]   PROTEINS IN DENATURING SOLVENTS - GEL EXCLUSION STUDIES [J].
DAVISON, PF .
SCIENCE, 1968, 161 (3844) :906-&
[9]   X-RAY CRYSTAL-STRUCTURES OF THE OXIDIZED AND REDUCED FORMS OF THE RUBREDOXIN FROM THE MARINE HYPERTHERMOPHILIC ARCHAEBACTERIUM PYROCOCCUS-FURIOSUS [J].
DAY, MW ;
HSU, BT ;
JOSHUATOR, L ;
PARK, JB ;
ZHOU, ZH ;
ADAMS, MWW ;
REES, DC .
PROTEIN SCIENCE, 1992, 1 (11) :1494-1507
[10]  
Eftink MR, 1995, METHOD ENZYMOL, V259, P487