The membrane-bound alpha-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylan

Abstract

The microbial degradation of xylan is a key biological process. Hardwood 4-O-methyl-D-glucuronoxylans are extensively decorated with 4-O-methyl-D-glucuronic acid, which is cleaved from the polysaccharides by alpha-glucuronidases. In this report we describe the primary structures of the alpha-glucuronidase from Cellvibrio mixtus (C. mixtus GlcA67A) and the alpha-glucuronidase from Pseudomonas cellulosa (P. cellulosa GlcA67A) and characterize P. cellulosa GlcA67A. The primary structures of C. mixtus GlcA67A and P. cellulosa GlcA67A, which are 76% identical, exhibit similarities with alpha-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad alpha-glucuronidase released 4-O-methyl-D-glucuronic acid from 4-O-methyl-D-glucuronoxylooligosaccharides but not from 4-O-methyl-D-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-O-methyl-D-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.

FIG. 1.

Physical map of agu67A locus. The positions of the cleavage sites for the following restriction enzymes are indicated: BamHI (B), EcoRI (R), EcoRV (E), HindIII (H), NcoII (N), PstI (P), SalI (S), SmaI (Sm), and SphI (Sp). The arrows show the extents and orientations of the genes encoding GlcA67A (agu67A), Xyn10D (xyn10D), and truncated DNA polymerase II (pol). Plasmid pTN4 was derived from recombinant phage, and pTN3, which encodes full-length GlcA67A, was a PCR product derived from pTN4. Plasmid pTN2, which encodes mature GlcA67A (residues 22 to 732), was derived from pTN3 by cloning the 2.1-kb NcoI-XhoI fragment into the expression vector pET32c. In pTN3 the α-glucuronidase gene was cloned into the pET expression vector pET28a, while pTN5 was derived by cloning a PCR product into the promoter probe vector pRG960SD. Glucuronidase activity refers to the presence of functional soluble enzyme activity in Escherichia coli strains carrying the plasmids.

FIG. 2.

Purification of GlcA67A. GlcA67A was purified as described in the text. The various fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using a 10% (wt/vol) polyacrylamide gel which contained the following samples: high-molecular-weight standards (lane 1); cell extract of E. coli containing pTN2, cultured in the absence (lane 2) and in the presence (lane 3) of isopropyl-β-d-thiogalactopyranoside (IPTG) (2); and protein purified by metal ion affinity chromatography (lane 4) and after enterokinase treatment (lane 5), anion-exchange chromatography (lane 6), and size exclusion chromatography (lane 7).