Substrate-Assisted Catalysis Unifies Two Families of Chitinolytic Enzymes

Abstract

Hen egg-white lysozyme has long been the paradigm for enzymatic glycosyl hydrolysis with retention of configuration, with a protonated carboxylic acid and a deprotonated carboxylate participating in general acid-base catalysis. In marked contrast, the retaining chitin degrading enzymes from glycosyl hydrolase families 18 and 20 all have a single glutamic acid as the catalytic acid but lack a nucleophile on the enzyme. Both families have a catalytic ( R) 8 -barrel domain in common. X-ray structures of three different chitinolytic enzymes complexed with substrates or inhibitors identify a retaining mechanism involving a protein acid and the carbonyl oxygen atom of the substrate's C2 N-acetyl group as the nucleophile. These studies unambiguously demonstrate the distortion of the sugar ring toward a sofa conformation, long postulated as being close to that of the transition state in glycosyl hydrolysis.

Structure of recombinant Ves v 2 at 2.0 Å resolution: structural analysis of an allergenic hyaluronidase from wasp venom

“…It is remarkable that the catalytic nucleophile is not an amino acid from the enzyme but instead the carbonyl O atom of the C-2 acetamido group of the substrate. This type of substrateassisted reaction is not unique; it has also been seen in glycoside hydrolase families 18 and 20 (Tews et al, 1997). Cocrystallization of Api m 2 with a substrate analogue mapped out some substrate-binding subsites and identified the active site of the enzyme (Markovic Housley et al, 2000).…”

Substrate positioning in chitinase A, a processive chito-biohydrolase fromSerratia marcescens

“…Such binding would be non-productive because of the involvement of the acetamidogroup of the -1 sugar in catalysis, [30]. Apparently, formation of non-productive complexes is promoted by removal of Trp 167 , since substrates that can be cleaved by the wild-type enzyme remain un-cleaved.…”

Functional Analysis of the Chitin-binding Domain of a Family 19 Chitinase fromStreptomyces griseusHUT6037: Substrate-binding Affinity andcis-Dominant Increase of Antifungal Function

“…3,4) Moreover, they use diŠerent catalytic mechanisms. Family 18 chitinases do substrate-assisted catalysis, 5,6) and family 19 chitinases use the general acid-and-base mechanism. 4,7) The family 18 chitinases are ubiquitous, but the family 19 enzymes have been identiˆed mostly in plants.…”