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

• Published in: Biochemistry (Easton) 2011-06, Vol.50 (25), p.5693-5703
• Main Authors: Zakariassen, Henrik, Hansen, Mona Cecilie, Jøranli, Maje, Eijsink, Vincent G. H, Sørlie, Morten
• Format: Article
• Language: English
• Subjects: Asparagine - genetics; Aspartic Acid - genetics; Bacterial Proteins - chemical synthesis; Bacterial Proteins - genetics; Catalytic Domain - genetics; Chitinases - chemical synthesis; Chitinases - classification; Chitinases - genetics; Enzyme Stability - genetics; Glycosylation; Hydrolysis; Multigene Family - genetics; Mutagenesis, Site-Directed; Oligosaccharides - chemistry; Oligosaccharides - genetics; Point Mutation; Serratia marcescens - enzymology; Serratia marcescens - genetics; Substrate Specificity - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi2002532

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 Asp313/142 to Ala or the mutation of Asp311/140 to either Asn or Ala had no or much smaller effects on transglycosylating activity. Mutation of Phe396 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.