Identifying the catalytic acid/base in GH29 α-l-fucosidase subfamilies

TY - JOUR

T1 - Identifying the catalytic acid/base in GH29 α-l-fucosidase subfamilies

AU - Shaikh, F.A.

AU - Lammerts Van Bueren, A.

AU - Davies, G.J.

AU - Withers, S.G.

PY - 2013/8/27

Y1 - 2013/8/27

N2 - While the catalytic nucleophile in the configuration-retaining α-l-fucosidases from family GH29 is fully conserved with respect to sequence, there is no fully sequence-conserved acid/base residue candidate across the family. X-ray crystallographic studies and kinetic characterizations have allowed the identification of this residue in a few cases, and a recent combination of phylogenetic tree analyses with substrate specificity data has allowed the division of GH29 enzymes into two subfamilies, A and B, allowing the probable assignment of these residues. Here, we perform detailed kinetic and mechanistic characterizations of the corresponding alanine mutants and other candidates. Through comparison of kinetic parameters obtained for the hydrolysis of fucosyl substrates with activated leaving groups by these mutants with those of the corresponding wild-type enzymes, in conjunction with the demonstration of azide rescue, we largely confirm the acid/base residue predictions for the GH29 fucosidases from the two subfamilies.

AB - While the catalytic nucleophile in the configuration-retaining α-l-fucosidases from family GH29 is fully conserved with respect to sequence, there is no fully sequence-conserved acid/base residue candidate across the family. X-ray crystallographic studies and kinetic characterizations have allowed the identification of this residue in a few cases, and a recent combination of phylogenetic tree analyses with substrate specificity data has allowed the division of GH29 enzymes into two subfamilies, A and B, allowing the probable assignment of these residues. Here, we perform detailed kinetic and mechanistic characterizations of the corresponding alanine mutants and other candidates. Through comparison of kinetic parameters obtained for the hydrolysis of fucosyl substrates with activated leaving groups by these mutants with those of the corresponding wild-type enzymes, in conjunction with the demonstration of azide rescue, we largely confirm the acid/base residue predictions for the GH29 fucosidases from the two subfamilies.

UR - http://www.scopus.com/inward/record.url?scp=84883250679&partnerID=8YFLogxK

U2 - 10.1021/bi400183q

DO - 10.1021/bi400183q

M3 - Article

AN - SCOPUS:84883250679

SN - 0006-2960

VL - 52

SP - 5857

EP - 5864

JO - Biochemistry

JF - Biochemistry

IS - 34

ER -