Substrate Specificity, Substrate Channeling, and Allostery in BphJ: An Acylating Aldehyde Dehydrogenase Associated with the Pyruvate Aldolase Bphl

被引:13
作者
Baker, Perrin [1 ]
Carere, Jason [1 ]
Seah, Stephen Y. K. [1 ]
机构
[1] Univ Guelph, Dept Mol & Cellular Biol, Guelph, ON N1G 2W1, Canada
基金
加拿大自然科学与工程研究理事会;
关键词
BETA-SEMIALDEHYDE DEHYDROGENASE; SITE-DIRECTED MUTAGENESIS; COENZYME-A; STREPTOCOCCUS-MUTANS; CATALYTIC MECHANISM; BACILLUS-SUBTILIS; CRYSTAL-STRUCTURE; D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE; GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE; KINETIC MECHANISM;
D O I
10.1021/bi300407y
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
BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-beta-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by >= 3-fold in the presence of NADH and >= 4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling.
引用
收藏
页码:4558 / 4567
页数:10
相关论文
共 52 条
[1]   Protein-Protein Interactions and Substrate Channeling in Orthologous and Chimeric Aldolase-Dehydrogenase Complexes [J].
Baker, Perrin ;
Hillis, Colleen ;
Carere, Jason ;
Seah, Stephen Y. K. .
BIOCHEMISTRY, 2012, 51 (09) :1942-1952
[2]   Rational Design of Stereoselectivity in the Class II Pyruvate Aldolase Bphl [J].
Baker, Perrin ;
Seah, Stephen Y. K. .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2012, 134 (01) :507-513
[3]   Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, Bphl [J].
Baker, Perrin ;
Carere, Jason ;
Seah, Stephen Y. K. .
BIOCHEMISTRY, 2011, 50 (17) :3559-3569
[4]   Characterization of an Aldolase-Dehydrogenase Complex That Exhibits Substrate Channeling in the Polychlorinated Biphenyls Degradation Pathway [J].
Baker, Perrin ;
Pan, Dan ;
Carere, Jason ;
Rossi, Adam ;
Wang, Weijun ;
Seah, Stephen Y. K. .
BIOCHEMISTRY, 2009, 48 (27) :6551-6558
[5]   Critical catalytic functional groups in the mechanism of aspartate-β-semialdehyde dehydrogenase [J].
Blanco, J ;
Moore, RA ;
Faehnle, CR ;
Viola, RE .
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, 2004, 60 :1808-1815
[6]   The role of substrate-binding groups in the mechanism of aspartate-β-semialdehyde dehydrogenase [J].
Blanco, J ;
Moore, RA ;
Faehnle, CR ;
Coe, DM ;
Viola, RE .
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, 2004, 60 :1388-1395
[7]   Capture of an intermediate in the catalytic cycle of L-aspartate-β-semialdehyde dehydrogenase [J].
Blanco, J ;
Moore, RA ;
Viola, RE .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2003, 100 (22) :12613-12617
[8]   A structural basis for the mechanism of aspartate-β-semialdehyde dehydrogenase from Vibrio cholerae [J].
Blanco, J ;
Moore, RA ;
Kabaleeswaran, V ;
Viola, RE .
PROTEIN SCIENCE, 2003, 12 (01) :27-33
[9]  
BRADBURY SL, 1971, J BIOL CHEM, V246, P1834
[10]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3