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

Abstract

<jats:p>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 <jats:italic>Serratia marcescens</jats:italic> (<jats:italic>Sm</jats:italic>ChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of <jats:italic>Sm</jats:italic>ChiA 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 <jats:italic>Sm</jats:italic>ChiA 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 <jats:italic>Sm</jats:italic>ChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for <jats:italic>Sm</jats:italic>ChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of <jats:italic>Sm</jats:italic>ChiA mutants.</jats:p>

Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases

“…This difference is one of the reasons that CfCel6B showed a lower K m value than TrCel6A. However, quantitative comparison is not easy, because direct measurement of the k on for productive binding was difficult because of the low frame rate and localization precisions, similar to the case for our recent single-molecule fluorescence imaging of chitinase A from Serratia marcescens ( 21 ).…”

Overexpression of Chitinase A Gene from Serratia marcescens in Bacillus subtilis and Characterization of Enhanced Chitinolytic Activity

“…Brazilian Archives of Biology and Technology. Vol.63: e20200061, 2020 www.scielo.br/babt walls of some fungi [1,2]. Water-soluble, degraded form of chitin is valuable both for biomass conversion and biotechnological applications [3].…”

“…Serratia marcescens is one of the chitinase over-producer bacteria [2,18,19]. There are some studies to ameliorate the characteristics of chitinase from S. marcescens.…”

Combined Approach to Engineer a Highly Active Mutant of Processive Chitinase Hydrolyzing Crystalline Chitin

“…Using single-molecule imaging analysis, we previously determined the kinetic parameters of elementary reaction steps and the chemomechanical coupling mechanism of Sm ChiA. 12 − 14 Recently, we further revealed that the F232W and F396W mutations at the beginning and end of the substrate-binding catalytic cleft decrease the dissociation rate constant of productively bound enzyme and increase processivity, and result in high catalytic activity. 14 , 15 Based on the multiple sequence alignment of the Sm ChiA and 257 Sm ChiA-like proteins, we also found that neither Phe232 nor Phe396 are predominant but tryptophan.…”

“… 12 − 14 Recently, we further revealed that the F232W and F396W mutations at the beginning and end of the substrate-binding catalytic cleft decrease the dissociation rate constant of productively bound enzyme and increase processivity, and result in high catalytic activity. 14 , 15 Based on the multiple sequence alignment of the Sm ChiA and 257 Sm ChiA-like proteins, we also found that neither Phe232 nor Phe396 are predominant but tryptophan. Our previous study strongly suggested that amino acid sequence of Sm ChiA is not optimized for high hydrolytic activity during the process of evolution in the nature.…”