Mutational Effects on Transglycosylating Activity of Family 18 Chitinases and Construction of a Hypertransglycosylating Mutant

Abstract

Enzymatic features that determine transglycosylating activity have been investigated through site-directed mutagenesis studies on two family 18 chitinases, ChiA and ChiB from Serratia marcescens, with inherently little transglycosylation activity. The activity was monitored for the natural substrate (GlcNAc)(4) using mass spectrometry and HPLC. Mutation of the middle Asp in the diagnostic DxDxE motif, which interacts with the catalytic Glu during the catalytic cycle, yielded the strongly transglycosylating mutants ChiA-D313N and ChiB-D142N, respectively. Mutation of the same Asp(313/142) to Ala or the mutation of Asp(311/140) to either Asn or Ala had no or much smaller effects on transglycosylating activity. Mutation of Phe(396) in the +2 subsite of ChiA-D313N to Trp led to a severalfold increase in transglycosylation rate while replacement of aromatic residues with Ala in the aglycon (sugar acceptor-binding) subsites of ChiA-D313N and ChiB-D142N led to a clear reduction in transglycosylating activity. Taken together, these results show that the transglycosylation properties of family 18 chitinases may be manipulated by mutations that affect the configuration of the catalytic machinery and the affinity for sugar acceptors. The hypertransglycosylating mutant ChiA-D313N-F396W may find applications for synthetic purposes.

Analysis of productive binding modes in the human chitotriosidase

“…Previous studies have indicated that HCHT has high transglycosylation activity that may be observed without applying typical ''transglycosylation conditions'' (high substrate and enzyme concentrations) [12]. The high transglycosylation activity of HCHT further supports the idea that this enzyme has relatively strong binding affinities in the positive subsites as high affinity for sugar acceptors is known to promote transglycosylation activity in family 18 chitinases [13,32].…”

“…Endo-activity would benefit from an extended substrate binding cleft with more than two subsites on each site of the catalytic center where cleavage takes place. High oligosaccharide affinity in multiple positive subsites have been shown to be beneficial for transglycosylation activity [13]. While the structures of AMCase [7] and HCHT [14] are known, there are no structures of complexes with longer ligands that could provide comprehensive insight into the subsite structure of the enzymes.…”

Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568

“…Thus, the amino acid residues responsible for acceptor binding appear to be important for TG activity. Mutation of the middle Asp in the diagnostic DxDxE motif, which interacts with the catalytic Glu during the catalytic cycle, yielded strong TG mutants (50). Correlation between a lower K m and increased TG was observed for the ␤-mannanase Man5A from the blue mussel Mytilus edulis (19), endo-␤-N-acetylglucosaminidase from Mucor hiemalis (39), and chitinase A from Vibrio carchariae (33).…”

Transglycosylation by Chitinase D from Serratia proteamaculans Improved through Altered Substrate Interactions

“…Hydrolysis occurs when a water molecule attacks the glycosyl-enzyme intermediate leading to the release of a hydrolyzed sugar. Alternatively, TG occurs when a carbohydrate molecule outcompetes with the water molecule (14).…”

“…Mutation of Trp-167 at the Ϫ3 subsite in chitinase A of Serratia marcescens (15) and a chemical modification of hen egg white lysozyme at Asp-101 and Trp-62 at Ϫ4 and Ϫ2 subsites, respectively, enhanced the TG activity (16). The variants of Asp-140/311 and Asp-142/313, analogous residues in ChiB and ChiA from S. marcescens, respectively, showed improved TG activity (14). Similarly, the aromatic side chains of Phe-166 and Trp-197 in class V chitinase from Cycas revoluta were important for the TG acceptor binding than for the substrate binding (17).…”