Synthesis of long-chain chitooligosaccharides by a hypertransglycosylating processive endochitinase of Serratia proteamaculans 568.
We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40 °C, where it showed a K(m) of 83 mg ml(-1), a k(cat) of 3.9 × 10(2) h(-1), and a k(cat)/K(m) of 4.7 h mg(-1) ml(-1) on colloidal chitin. On chitobiose, the K(m), k(cat), and k(cat)/K(m) were 203 μM, 1.3 × 10(2) h(-1), and 0.62 h(-1) μM(-1), respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase.
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Pallinti
P
Purushotham
Appa Rao
AR
Podile
D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D008958:Models, Molecular; D009844:Oligosaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012705:Serratia / Q000201:enzymology*; D013696:Temperature
D009844:Oligosaccharides; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases