The molecular, biochemical and biophysical characterization of a Thermotoga neapolitana N-acetylglucosaminidase.

摘要

Chitin is the most abundant nitrogen bearing organic compound in nature, and is the structural support of insect exoskeletons, crustacean shells and fungal cell walls. Its hydrolysis has both ecological and applied significance, for instance, yielding an important carbon and nitrogen source for marine bacteria. They degrade chitin by a multiple enzyme system generally consisting of endochitinases and N-acetylglucosaminidases.; Thermotoga neapolitana, a marine hyperthermophilic bacterium, is capable of metabolizing a variety of carbohydrates. A genomic library of Thermotoga neapolitana was screened for activity against chitin analogous substrates. A clone 74C11 exhibiting N-acetylglucosaminidase activity, cbsA, was identified. The cbsA gene codes for 467 amino acids, with a high homology to N-acetylglucosaminidases belonging to family 3 glycosyl hydrolases. The recombinant cbsA was cloned in Escherichia cola in an expression vector that facilitated affinity purification. The 2-step purification resulted in a final eighty-fold purification with a 49% yield. The enzyme had a molecular mass of 50kDa with a pI of 5.5. CbsA was thermostable with a half-life of 240 min at 85°C and pH optimum of 8.0. CbsA had a broad substrate specificity with activity against chitobiose to chitohexose. Kinetic studies revealed chitobiose as its preferred substrate. The enzyme was exo-acting and non-processive.; The three-dimensional structure of CbsA was modeled based on the crystal structure of family 3 barley β-glucan-glucohydrolase, in order to elucidate the structural basis for its broad substrate specificity. The CbsA model has an N-terminal (αβ)₈ TIM barrel domain and a truncated C-terminal (αβ)₆ domain connected by a 16 amino acid linker. The active site pocket lies in the cleft between the two domains of the modeled CbsA. It is shaped like a coin slot and is approximately 20 +/− 3 Å deep. The catalytic nucleophile in the CbsA model was putatively identified as an aspartate, D245, and the catalytic acid was putatively identified as a glutamate, E65. Both catalytic residues mapped in the active site pocket.; Molecular, biochemical and biophysical characterization of T. neapolitana CbsA in conjunction with directed evolution approaches will form the basis of the next generation of experiments aimed at improving the properties of this enzyme.