TY - JOUR
T1 - Structure-based engineering of glucose specificity in a family 10 xylanase from Streptomyces olivaceoviridis E-86
AU - Ichinose, Hitomi
AU - Diertavitian, Shaghik W Sarkis
AU - Fujimoto, Zui
AU - Kuno, Atsushi
AU - Lo Leggio, Leila
AU - Kaneko, Satoshi
N1 - Biomass-derived pentoses
PY - 2012/3
Y1 - 2012/3
N2 - Substrate specificity is one of the most important functional property of enzymes. We use family 10 xylanase from Streptomyces olivaceoviridis as a model for substrate specificity of glycoside hydrolases. Seven variants were initially designed to change the preference from xylose to glucose at substrate binding subsites -2 and -1. The known mobility of Trp at the -1 subsite and the influence of its environment, which is different in subset 1 and subset 2 family 10 enzymes, were taken into account in variant design. Q88A/R275A had the best ratio of p-nitrophenyl cellobioside vs p-nitrophenyl xylobioside hydrolyzing activity in the first series of variants. The crystal structure shows a movement of Trp274 compared to the native, as a result of loss of interaction with the long side chain of Arg275. The movement creates extra space for the hydroxymethyl of glucose, resulting in improved K m on glucose derived substrates, while the negative effect on k cat is compensated by the Q88A mutation, which also contributes to a further reduction of K m. Further mutagenesis based on the Q88A/R275A variant resulted in 5.2 times improvement compared to the wild-type p-nitrophenyl cellobioside hydrolyzing activity, which is the best improvement obtained so far for an engineered xylanase.
AB - Substrate specificity is one of the most important functional property of enzymes. We use family 10 xylanase from Streptomyces olivaceoviridis as a model for substrate specificity of glycoside hydrolases. Seven variants were initially designed to change the preference from xylose to glucose at substrate binding subsites -2 and -1. The known mobility of Trp at the -1 subsite and the influence of its environment, which is different in subset 1 and subset 2 family 10 enzymes, were taken into account in variant design. Q88A/R275A had the best ratio of p-nitrophenyl cellobioside vs p-nitrophenyl xylobioside hydrolyzing activity in the first series of variants. The crystal structure shows a movement of Trp274 compared to the native, as a result of loss of interaction with the long side chain of Arg275. The movement creates extra space for the hydroxymethyl of glucose, resulting in improved K m on glucose derived substrates, while the negative effect on k cat is compensated by the Q88A mutation, which also contributes to a further reduction of K m. Further mutagenesis based on the Q88A/R275A variant resulted in 5.2 times improvement compared to the wild-type p-nitrophenyl cellobioside hydrolyzing activity, which is the best improvement obtained so far for an engineered xylanase.
U2 - 10.1016/j.procbio.2011.06.002
DO - 10.1016/j.procbio.2011.06.002
M3 - Journal article
SN - 1359-5113
VL - 47
SP - 358
EP - 365
JO - Process Biochemistry
JF - Process Biochemistry
IS - 3
ER -