The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 Å resolution

被引:101
作者
Sandgren, M
Shaw, A
Ropp, TH
Bott, SWR
Cameron, AD
Ståhlberg, J
Mitchinson, C
Jones, TA
机构
[1] Uppsala Univ, Dept Cell & Mol Biol, SE-75124 Uppsala, Sweden
[2] Uppsala Univ, Dept Mol Biol, Ctr Biomed, SE-75124 Uppsala, Sweden
[3] Genencor Int, Palo Alto, CA 94304 USA
关键词
protein structure; cellulase; cellulose; endoglucanase; Trichoderma reesei;
D O I
10.1006/jmbi.2001.4583
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
We present the three-dimensional structure of Trichoderma reesei endoglucanase 3 (Cell2A), a small, 218 amino acid residue (24.5 kDa), neutral pi, glycoside hydrolase family 12 cellulase that lacks a cellulose-binding module. The structure has been determined using X-ray crystallography and refined to 1.9 Angstrom resolution. The asymmetric unit consists of six noncrystallographic symmetry-related molecules that were exploited to improve initial multiple isomorphous replacement phasing, and subsequent structure refinement. The enzyme contains one disulfide bridge and is glycosylated at Asp164 by a single N-acetyl glucosamine residue. The protein has the expected fold for a glycoside hydrolase clan-C family 12 enzyme. It contains two beta -sheets, of six and nine strands, packed on top of one another, and one alpha -helix. The concave surface of the nine-stranded beta -sheet forms a large substrate-binding groove in which the active-site residues are located. In the active site, we find a carboxylic acid trio, similar to that of glycoside hydrolase families 7 and 16. The strictly conserved Asp99 hydrogen bonds to the nucleophile, the invariant Glu116. The binding crevice is lined with both aromatic and polar amino acid side-chains which may play a role in substrate binding. The structure of the fungal family 12 enzyme presented here allows a complete structural characterization of the glycoside hydrolase-C clan. (C) 2001 Academic Press.
引用
收藏
页码:295 / 310
页数:16
相关论文
共 56 条
[1]  
[Anonymous], 1991, P CCP4 STUD WEEK IS
[2]   THE DEVELOPMENT OF GENE-EXPRESSION SYSTEMS FOR FILAMENTOUS FUNGI [J].
BERKA, RM ;
BARNETT, CC .
BIOTECHNOLOGY ADVANCES, 1989, 7 (02) :127-154
[3]   PROTEIN DATA BANK - COMPUTER-BASED ARCHIVAL FILE FOR MACROMOLECULAR STRUCTURES [J].
BERNSTEIN, FC ;
KOETZLE, TF ;
WILLIAMS, GJB ;
MEYER, EF ;
BRICE, MD ;
RODGERS, JR ;
KENNARD, O ;
SHIMANOUCHI, T ;
TASUMI, M .
JOURNAL OF MOLECULAR BIOLOGY, 1977, 112 (03) :535-542
[4]  
BOWER B, 1998, CARBOHYDRASES TRICHO, P327
[5]   FREE R-VALUE - A NOVEL STATISTICAL QUANTITY FOR ASSESSING THE ACCURACY OF CRYSTAL-STRUCTURES [J].
BRUNGER, AT .
NATURE, 1992, 355 (6359) :472-475
[6]   Crystallography & NMR system:: A new software suite for macromolecular structure determination [J].
Brunger, AT ;
Adams, PD ;
Clore, GM ;
DeLano, WL ;
Gros, P ;
Grosse-Kunstleve, RW ;
Jiang, JS ;
Kuszewski, J ;
Nilges, M ;
Pannu, NS ;
Read, RJ ;
Rice, LM ;
Simonson, T ;
Warren, GL .
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1998, 54 :905-921
[7]   Miscellaneous algorithms for density modification [J].
Cowtan, K ;
Main, P .
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, 1998, 54 :487-493
[8]   Nomenclature for sugar-binding subsites in glycosyl hydrolases [J].
Davies, GJ ;
Wilson, KS ;
Henrissat, B .
BIOCHEMICAL JOURNAL, 1997, 321 :557-559
[9]   THE 3-DIMENSIONAL CRYSTAL-STRUCTURE OF THE CATALYTIC CORE OF CELLOBIOHYDROLASE-I FROM TRICHODERMA-REESEI [J].
DIVNE, C ;
STAHLBERG, J ;
REINIKAINEN, T ;
RUOHONEN, L ;
PETTERSSON, G ;
KNOWLES, JKC ;
TEERI, TT ;
JONES, TA .
SCIENCE, 1994, 265 (5171) :524-528
[10]  
Gottschalk G., 1988, Biochemistry and genetics of cellulose degradation, P3