The C-terminal module of Chi1 from Aeromonas caviae CB101 has a function in substrate binding and hydrolysis.
The chitinase gene chi1 of Aeromonas caviae CB101 encodes an 865-amino-acid protein (with signal peptide) composed of four domains named from the N-terminal as an all-beta-sheet domain ChiN, a triosephosphate isomerase (TIM) catalytic domain, a function-unknown A region, and a putative chitin-binding domain (ChBD) composed of two repeated sequences. The N-terminal 563-amino-acid segment of Chi1 (Chi1DeltaADeltaChBD) shares 74% identity with ChiA of Serratia marcescens. By the homology modeling method, the three-dimensional (3D) structure of Chi1DeltaADeltaChBD was constructed. It fit the structure of ChiA very well. To understand fully the function of the C-terminal module of Chi1 (from 564 to 865 amino acids), two different C-terminal truncates, Chi1DeltaChBD and Chi1DeltaADeltaChBD, were constructed, based on polymerase chain reaction (PCR). Comparison studies of the substrate binding, hydrolysis capacity, and specificity among Chi1 and its two truncates showed that the C-terminal putative ChBD contributed to the insoluble substrate-protein binding and hydrolysis; the A region did not have any function in the insoluble substrate-protein binding, but it did have a role in the chitin hydrolysis: Deletion of the A region caused the enzyme to lose 30-40% of its activity toward amorphous colloidal chitin and soluble chitin, and around 50% toward p-nitrophenyl (pNP)-chitobiose pNP-chitotriose, and its activity toward low-molecular-weight chitooligomers (GlcNAc)3-6 also dropped, as shown by analysis of its digestion processes. This is the first clear demonstration that a domain or segment without a function in insoluble substrate-chitinase binding has a role in the digestion of a broad range of chitin substrates, including low-molecular-weight chitin oligomers. The reaction mode of Chi1 is also described and discussed.
Wang
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FP
Key Laboratory of Marine Biogenetic Resources, State Oceanic Administration, and Third Institute of Oceanography, State Oceanic Administration, China.
Li
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Zhou
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Y
Li
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MG
Xiao
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D000333:Aeromonas / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases