Biochemical and genetic characterization of ChiA, the major enzyme component for the solubilization of chitin by Cellulomonas uda.
Cellulomonas uda efficiently solubilized chitinous substrates with a simple chitinase system composed of an endochitinase, designated ChiA, which hydrolyzed insoluble substrates into long-chain chitooligosaccharides, and an as yet uncharacterized exochitinase activity. ChiA, isolated from culture supernatant fluids, was found to be a glycosylated endochitinase with an apparent molecular mass of approximately 70 kDa and pI of 8.5. The gene encoding ChiA was cloned in Escherichia coli and sequenced, revealing an open reading frame of 1,716 bp encoding a 571-amino-acid protein with a predicted molecular mass of 59.2 kDa. The region upstream of chiA included a conserved -35 hexamer flanked by two direct repeats analogous to those found in many Streptomyces chitinase promoters, and thought to function as binding sequences for regulatory proteins. Analysis of the deduced amino acid sequence showed a modular protein consisting of a signal peptide at its N terminus, a family 2 carbohydrate-binding module (CBM2) that was closely related to the substrate-binding domains of glycosyl hydrolases from distantly related bacteria, and a family 18 glycosyl hydrolase catalytic module related to Streptomyces chitinases. In contrast to the fibronectin type III domains of Streptomyces chitinases, the linker region between modules in ChiA consisted of a long proline- and threonine-rich module, thought to contribute to the glycosylation and flexibility of the mature protein.
Reguera
Gemma
G
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Leschine
Susan B
SB
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D023061:Gene Order; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006023:Glycoproteins; D002686:Chitin; D002688:Chitinases; C094590:chitinase ChiA