Structure and function analysis of Pseudomonas plant cell wall hydrolases.
Hydrolysis of the major structural polysaccharides of plant cell walls by the aerobic soil bacterium Pseudomonas fluorescens subsp. cellulosa is attributable to the production of multiple extracellular cellulase and hemicellulase enzymes, which are the products of distinct genes belonging to multigene families. Cloning and sequencing of individual genes, coupled with gene sectioning and functional analysis of the encoded proteins have provided a detailed picture of structure/function relationships and have established the cellulase-hemicellulase system of P. fluorescens subsp. cellulosa as a model for the plant cell wall degrading enzyme systems of aerobic cellulolytic bacteria. Cellulose- and xylan-degrading enzymes produced by the pseudomonad are typically modular in structure and contain catalytic and noncatalytic domains joined together by serine-rich linker sequences. The cellulases include a cellodextrinase; a beta-glucan glucohydrolase and multiple endoglucanases, containing catalytic domains belonging to glycosyl hydrolase families 5, 9, and 45; and cellulose-binding domains of families II and X, both of which are present in each enzyme. Endo-acting xylanases, with catalytic domains belonging to families 10 and 11, and accessory xylan-degrading enzymes produced by P. fluorescens subsp. cellulosa contain cellulose-binding domains of families II, X, and XI, which act by promoting close contact between the catalytic domain of the enzyme and its target substrate. A domain homologous with NodB from rhizobia, present in one xylanase, functions as a deacetylase. Mananase, arabinanase, and galactanase produced by the pseudomonad are single domain enzymes. Crystallographic studies, coupled with detailed kinetic analysis of mutant forms of the enzyme in which key residues have been altered by site-directed mutagenesis, have shown that xylanase A (family 10) has 8-fold alpha/beta barrel architecture, an extended substrate-binding cleft containing at least six xylose-binding pockets and a calcium-binding site that protects the enzyme from thermal inactivation, thermal unfolding, and attack by proteinases. Kinetic studies of mutant and wild-type forms of a mannanase and a galactanase from P. fluorescens subsp. cellulosa have enabled the catalytic mechanisms and key catalytic residues of these enzymes to be identified.
Hazlewood
G P
GP
Laboratory of Molecular Enzymology, Babraham Institute, Cambridge, United Kingdom.
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002473:Cell Wall / Q000737:chemistry; D002480:Cellulase / Q000737:chemistry; D002482:Cellulose / Q000032:analysis / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010944:Plants / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry
D011134:Polysaccharides; D002482:Cellulose; D006867:Hydrolases; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D002480:Cellulase