Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate

被引:17
作者
Voss, Jarrod E. [1 ,2 ]
Scally, Stephen W. [1 ,2 ]
Taylor, Nicole L. [1 ,2 ]
Dogovski, Con [1 ,2 ]
Alderton, Malcolm R. [3 ]
Hutton, Craig A. [2 ,4 ]
Gerrard, Juliet A. [5 ]
Parker, Michael W. [1 ,2 ,6 ]
Dobson, Renwick C. J. [1 ,2 ]
Perugini, Matthew A. [1 ,2 ]
机构
[1] Univ Melbourne, Dept Biochem & Mol Biol, Parkville, Vic 3010, Australia
[2] Bio21 Mol Sci & Biotechnol Inst, Parkville, Vic 3010, Australia
[3] Def Sci & Technol Org, Human Protect & Performance Div, Port Melbourne, Vic 3207, Australia
[4] Univ Melbourne, Sch Chem, Parkville, Vic 3010, Australia
[5] Univ Canterbury, Sch Biol Sci, Christchurch 8020, New Zealand
[6] St Vincents Inst Med Res, Fitzroy, Vic 3065, Australia
来源
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS | 2009年 / 65卷
关键词
ESCHERICHIA-COLI; LYSINE BIOSYNTHESIS; CRYSTAL-STRUCTURE; 1ST SUBSTRATE; INHIBITION; RESOLUTION; CRYSTALLOGRAPHY;
D O I
10.1107/S1744309109000670
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 A are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme.
引用
收藏
页码:188 / 191
页数:4
相关论文
共 22 条
[11]   Dihydrodipicolinate synthase (DHDPS) from Escherichia coli displays partial mixed inhibition with respect to its first substrate, pyruvate [J].
Dobson, RCJ ;
Griffin, MDW ;
Roberts, SJ ;
Gerrard, JA .
BIOCHIMIE, 2004, 86 (4-5) :311-315
[12]   CHOOCH:: a program for deriving anomalous-scattering factors from X-ray fluorescence spectra [J].
Evans, G ;
Pettifer, RF .
JOURNAL OF APPLIED CRYSTALLOGRAPHY, 2001, 34 :82-86
[13]   ISOLATION AND CHARACTERIZATION OF DIHYDRODIPICOLINATE SYNTHASE FROM MAIZE [J].
FRISCH, DA ;
GENGENBACH, BG ;
TOMMEY, AM ;
SELLNER, JM ;
SOMERS, DA ;
MYERS, DE .
PLANT PHYSIOLOGY, 1991, 96 (02) :444-452
[14]   Structural and functional characterization of Staphylococcus aureus dihydrodipicolinate synthase [J].
Girish, Tavarekere S. ;
Sharma, Eshita ;
Gopal, B. .
FEBS LETTERS, 2008, 582 (19) :2923-2930
[15]   Inhibition of lysine biosynthesis: an evolving antibiotic strategy [J].
Hutton, Craig A. ;
Perugini, Matthew A. ;
Gerrard, Juliet A. .
MOLECULAR BIOSYSTEMS, 2007, 3 (07) :458-465
[16]   Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis [J].
Kefala, Georgia ;
Evans, Genevieve L. ;
Griffin, Michael D. W. ;
Devenish, Sean R. A. ;
Pearce, F. Grant ;
Perugini, Matthew A. ;
Gerrard, Juliet A. ;
Weiss, Manfred S. ;
Dobson, Renwick C. J. .
BIOCHEMICAL JOURNAL, 2008, 411 (02) :351-360
[17]   ESCHERICHIA-COLI DIHYDRODIPICOLINATE SYNTHASE - IDENTIFICATION OF THE ACTIVE-SITE AND CRYSTALLIZATION [J].
LABER, B ;
GOMISRUTH, FX ;
ROMAO, MJ ;
HUBER, R .
BIOCHEMICAL JOURNAL, 1992, 288 :691-695
[18]  
LESLIE AGW, 1991, CRYSTALLOGRAPHIC COM, V5, P50
[19]   Solving structures of protein complexes by molecular replacement with Phaser [J].
McCoy, Airlie J. .
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, 2007, 63 :32-41
[20]   Blu-Ice and the Distributed Control System:: software for data acquisition and instrument control at macromolecular crystallography beamlines [J].
McPhillips, TM ;
McPhillips, SE ;
Chiu, HJ ;
Cohen, AE ;
Deacon, AM ;
Ellis, PJ ;
Garman, E ;
Gonzalez, A ;
Sauter, NK ;
Phizackerley, RP ;
Soltis, SM ;
Kuhn, P .
JOURNAL OF SYNCHROTRON RADIATION, 2002, 9 :401-406