Novel β-N-acetylglucosaminidases from Vibrio harveyi 650: Cloning, expression, enzymatic properties, and subsite identification

<p>Abstract</p> <p>Background</p> <p>Since chitin is a highly abundant natural biopolymer, many attempts have been made to convert this insoluble polysaccharide into commercially valuable products using chitinases and <it>β</it>-<it>N</it>-acetylglucosaminidases (GlcNAcases). We have previously reported the structure and function of chitinase A from <it>Vibrio harveyi </it>650. This study t reports the identification of two GlcNAcases from the same organism and their detailed functional characterization.</p> <p>Results</p> <p>The genes encoding two new members of family-20 GlcNAcases were isolated from the genome of <it>V. harveyi </it>650, cloned and expressed at a high level in <it>E. coli</it>. <it>Vh</it>Nag1 has a molecular mass of 89 kDa and an optimum pH of 7.5, whereas <it>Vh</it>Nag2 has a molecular mass of 73 kDa and an optimum pH of 7.0. The recombinant GlcNAcases were found to hydrolyze all the natural substrates, <it>Vh</it>Nag2 being ten-fold more active than <it>Vh</it>Nag1. Product analysis by TLC and quantitative HPLC suggested that <it>Vh</it>Nag2 degraded chitooligosaccharides in a sequential manner, its highest activity being with chitotetraose. Kinetic modeling of the enzymic reaction revealed that binding at subsites (-2) and (+4) had unfavorable (positive) binding free energy changes and that the binding pocket of <it>Vh</it>Nag2 contains four GlcNAc binding subsites, designated (-1),(+1),(+2), and (+3).</p> <p>Conclusions</p> <p>Two novel GlcNAcases were identified as exolytic enzymes that degraded chitin oligosaccharides, releasing GlcNAc as the end product. In living cells, these intracellular enzymes may work after endolytic chitinases to complete chitin degradation. The availability of the two GlcNAcases, together with the previously-reported chitinase A from the same organism, suggests that a systematic development of the chitin-degrading enzymes may provide a valuable tool in commercial chitin bioconversion.</p

A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria

VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)2 across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)2, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)3–4]. (GlcNAc)4 partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp363, Asp365, and Trp513) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)2, indicating their significant contributions to sugar binding. The structure of the W513A–(GlcNAc)2 complex in a ‘half-open’ conformation unveiled the intermediary step of the (GlcNAc)2 translocation from the soluble CBP in the periplasm to the inner membrane–transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)2, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients

Structural basis for chitin acquisition by marine <i>Vibrio </i>species

Chitin degrading bacteria are important for marine ecosystems. Here the authors structurally and functionally characterize the Vibrio harveyi outer membrane diffusion channel chitoporin and give mechanistic insights into chito-oligosaccharide uptake

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

โครงการหนึ่งอาจารย์หนึ่งผลงาน ประจำปี 254

An endochitnase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis.

โครงการหนึ่งอาจารย์หนึ่งผลงา

Functional reconstitution,gene isolation and topology modeelling of porins from Burkholderia pseudomallei and B.thailandensis

ผลงานวิชาการคณาจารย์มหาวิทยาลัยเทคโนโลยีสุรนาร