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1.  A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding 
BMC Biochemistry  2013;14:23.
Background
Streptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42.
Results
Mutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity.
Conclusions
Arginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan.
doi:10.1186/1471-2091-14-23
PMCID: PMC3848431  PMID: 24041306
Chitosanase; Glycoside hydrolase family GH46; Substrate inhibition; Inverting mechanism; Enzyme-substrate interaction; Arginine
2.  Crystallization and preliminary X-ray diffraction analysis of a class V chitinase from Nicotiana tabacum  
N. tabacum class V chitinase has been crystallized. X-ray diffraction data were collected to 1.2 Å resolution using a synchrotron-radiation source.
The plant chitinases, which have been implicated in self-defence against pathogens, are divided into at least five classes (classes I, II, III, IV and V). Although the crystal structures of several plant chitinases have been solved, no crystal structure of a class V chitinase has been reported to date. Here, the crystallization of Nicotiana tabacum class V chitinase (NtChiV) using the vapour-diffusion method is reported. The NtChiV crystals diffracted to 1.2 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 62.4, b = 120.3, c = 51.9 Å. The asymmetric unit of the crystals is expected to contain one molecule.
doi:10.1107/S1744309110039060
PMCID: PMC2998363  PMID: 21139204
Nicotiana tabacum; defence proteins; class V chitinases
3.  Novel β-N-acetylglucosaminidases from Vibrio harveyi 650: Cloning, expression, enzymatic properties, and subsite identification 
BMC Biochemistry  2010;11:40.
Background
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 β-N-acetylglucosaminidases (GlcNAcases). We have previously reported the structure and function of chitinase A from Vibrio harveyi 650. This study t reports the identification of two GlcNAcases from the same organism and their detailed functional characterization.
Results
The genes encoding two new members of family-20 GlcNAcases were isolated from the genome of V. harveyi 650, cloned and expressed at a high level in E. coli. VhNag1 has a molecular mass of 89 kDa and an optimum pH of 7.5, whereas VhNag2 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, VhNag2 being ten-fold more active than VhNag1. Product analysis by TLC and quantitative HPLC suggested that VhNag2 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 VhNag2 contains four GlcNAc binding subsites, designated (-1),(+1),(+2), and (+3).
Conclusions
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.
doi:10.1186/1471-2091-11-40
PMCID: PMC2955587  PMID: 20920218

Results 1-3 (3)