Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from Serratia marcescens

Akasit Visootsat,Akihiko Nakamura,Paul Vignon,Hiroki Watanabe,Takayuki Uchihashi,Ryota Iino

doi:10.1074/jbc.ra119.012078

Abstract

Chitin degradation is important for biomass conversion and has potential applications for agriculture, biotechnology, and the pharmaceutical industry. Chitinase A from the Gram-negative bacterium Serratia marcescens (SmChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of SmChiA against crystalline chitin was found to increase after the tryptophan substitution of two phenylalanine residues (F232W and F396W), located at the entrance and exit of the substrate binding cleft of the catalytic domain, respectively. However, the mechanism underlying this high catalytic activity remains elusive. In this study, single-molecule fluorescence imaging and high-speed atomic force microscopy were applied to understand the mechanism of this high-catalytic-activity mutant. A reaction scheme including processive catalysis was used to reproduce the properties of SmChiA WT and F232W/F396W, in which all of the kinetic parameters were experimentally determined. High activity of F232W/F396W mutant was caused by a high processivity and a low dissociation rate constant after productive binding. The turnover numbers for both WT and F232W/F396W, determined by the biochemical analysis, were well-replicated using the kinetic parameters obtained from single-molecule imaging analysis, indicating the validity of the reaction scheme. Furthermore, alignment of amino acid sequences of 258 SmChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for SmChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of SmChiA mutants.

Highlights

Our biochemical analysis confirmed that the F232W/F396W mutant of SmChiA has a higher hydrolytic activity than the WT, as shown previously [38] (Fig. 2)
We found that F232W/ F396W showed higher hydrolytic activity than WT at all chitin concentrations ranging from 0.063 to 6 mg/ml
Hydrolytic activities were slightly inhibited for both WT and F232W/ F396W at chitin concentrations higher than 1 mg/ml and could not be fitted using the Michaelis–Menten equation (Fig. 2A)

Summary

To whom correspondence should be addressed

Processive cellulases and chitinases share the similar feature of a long and deep substrate-binding cleft and substrate-binding surface, which contain aromatic amino acid residues [20, 21, 25] These aromatic residues play an important role in the carbohydrate-protein interaction by which hydrophobic stacking (CH-␲ interaction) is formed between the aromatic side chain and sugar ring. Studies on processivity of the cellulases and chitinases using biochemical methods, such as the fluorescence labeling of the substrate [30, 31] and 14C-labeled chitin [32], or via the use of biosensors [33] have been performed extensively.

Results

Discussion

Experimental procedures