The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains.
A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in pUC18 and Escherichia coli recombinants expressing 4-methylumbelliferyl beta-D-cellobioside-hydrolysing activity (MUCase) were isolated. Enzyme produced by MUCase-positive clones did not hydrolyse either cellobiose or cellotriose but converted cellotetraose into cellobiose and cleaved cellopentaose and cellohexaose, producing a mixture of cellobiose and cellotriose. There was no activity against CM-cellulose, insoluble cellulose or xylan. On this basis, the enzyme is identified as an endo-acting cellodextrinase and is designated cellodextrinase C (CELC). Nucleotide sequencing of the gene (celC) which directs the synthesis of CELC revealed an open reading frame of 2153 bp, encoding a protein of Mr 80,189. The deduced primary sequence of CELC was confirmed by the Mr of purified CELC (77,000) and by the experimentally determined N-terminus of the enzyme which was identical with residues 38-47 of the translated sequence. The N-terminal region of CELC showed strong homology with endoglucanase, xylanases and an arabinofuranosidase of Ps. fluorescens subsp. cellulosa; homologous sequences included highly conserved serine-rich regions. Full-length CELC bound tightly to crystalline cellulose. Truncated forms of celC from which the DNA sequence encoding the conserved domain had been deleted, directed the synthesis of a functional cellodextrinase that did not bind to crystalline cellulose. This is consistent with the N-terminal region of CELC comprising a non-catalytic cellulose-binding domain which is distinct from the catalytic domain. The role of the cellulose-binding region is discussed.
Ferreira
L M
LM
Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, U.K.
Hazlewood
G P
GP
Barker
P J
PJ
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; C066565:ced1 protein, Butyrivibrio fibrisolvens; D002480:Cellulase