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Abstract
Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain. Here, we analyse the mechanism underlying unidirectional movement of Serratia marcescens chitinase A (SmChiA) using high-precision single-molecule imaging, X-ray crystallography, and all-atom molecular dynamics simulation. SmChiA shows fast unidirectional movement of ~50 nm s−1 with 1 nm forward and backward steps, consistent with the length of reaction product chitobiose. Analysis of the kinetic isotope effect reveals fast substrate-assisted catalysis with time constant of ~3 ms. Decrystallization of the single chitin chain from crystal surface is the rate-limiting step of movement with time constant of ~17 ms, achieved by binding free energy at the product-binding site of SmChiA. Our results demonstrate that SmChiA operates as a burnt-bridge Brownian ratchet wherein the Brownian motion along the single chitin chain is rectified forward by substrate-assisted catalysis.
Highlights
Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain
To achieve localisation precision and temporal resolution required for the detection of 1 nm steps of processive chitinase, we labelled the most studied chitinase A from a bacterium Serratia marcescens (SmChiA) with a 40 nm AuNP and observed scattering images using total internal reflection dark-field microscopy (TIRDFM; Fig. 1b)[27,28,29]
We analysed signal intensity, velocity, and run length of AuNP-labelled Serratia marcescens chitinase A (SmChiA) molecules moving on the crystalline chitin
Summary
Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain. Decrystallization of the single chitin chain from crystal surface is the rate-limiting step of movement with time constant of ~17 ms, achieved by binding free energy at the productbinding site of SmChiA. In addition to the processive movements, single-molecule imaging has revealed that chitinase moves and, produces disaccharides ten times faster than cellulase does[4,5], the physical and chemical stabilities of crystalline cellulose and chitin are similar[24,25]. During the processive movement on crystalline chitin surface, chitinase keeps binding with single chitin chain in the catalytic cleft and repeats chemical and mechanical steps (Fig. 1b). Decrystallisation of single chitin chain is the rate-limiting step of movement, achieved by binding free energy at the product-binding site, and verified by X-ray crystallographic structural analysis and all-atom molecular dynamics (MD) simulations of the intermediate structures during sliding movement. The strategy evolved by chitinase can be applied to design fast-moving artificial molecular motors such as DNA walkers[3]
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