Effect of single active-site cleft mutation on product specificity in a thermostable bacterial cellulase.
Mutation of a single active-site cleft tyrosyl residue to a glycyl residue significantly changes the mixture of products released from phosphoric acid-swollen cellulose (PSC) by EIcd, the catalytic domain of the endoglucanase-I from Acidothermus cellulolyticus. The percentage of glucose in the product stream is almost 40% greater for the Y245G mutant (and for an additional double mutant, Y245G/Q204A) than for the wild type enzyme. Comparisons of results for digestion PSC and of pretreated yellow poplar suggest that the observed shifts in product specificity are connected to the hydrolysis of a more easily digestible fraction of both substrates. A model is presented that relates the changes in product specificity to a mutation-driven shift in indexing of the polymeric substrate along the extended binding-site cleft.
Rignall
Tauna R
TR
Biotechnology for Fuels and Chemicals Division, National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, USA.
Baker
John O
JO
McCarter
Suzanne L
SL
Adney
William S
WS
Vinzant
Todd B
TB
Decker
Stephen R
SR
Himmel
Michael E
ME
D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006358:Hot Temperature; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D011994:Recombinant Proteins; C056374:endoglucanase 1; D002480:Cellulase