Biochemical and structural characterization of a novel 4-O-α-l-rhamnosyl-β-d-glucuronidase from Fusarium oxysporum

In this study, we have isolated the novel enzyme 4-O-alpha-l-rhamnosyl-beta-d-glucuronidase (FoBGlcA), which releases alpha-l-rhamnosyl (1 -> 4) glucuronic acid from gum arabic (GA), from Fusarium oxysporum 12S culture supernatant, and for the first time report an enzyme with such catalytic activity. The gene encoding FoBGlcA was cloned and expressed in Pichia pastoris. When GA was subjected to the recombinant enzyme, > 95% of the l-rhamnose (Rha) and d-glucuronic acid in the substrate were released, which indicates that almost all Rha binds to the glucuronic acid at the end of the GA side chains. The crystal structure of FoBGlcA was determined using a single-wavelength anomalous dispersion at 1.51 angstrom resolution. FoBGlcA consisted of an N-terminal (beta/alpha)(8)-barrel domain and a C-terminal antiparallel beta-sheet domain. This configuration is characteristic of glycoside hydrolase (GH) family 79 proteins. A structural similarity search showed that FoBGlcA mostly resembled GH79 beta-d-glucuronidase (AcGlcA79A) of Acidobacterium capsulatum; however, the root-mean-square deviation value was 3.2 angstrom, indicating that FoBGlcA has a high structural divergence. FoBGlcA had a low sequence identity with AcGlcA79A (19%) and differed from other GH79 beta-glucuronidases. The structures of FoBGlcA and AcGlcA79A also differed in terms of the loop structure location near subsite -2 of their catalytic sites, which may account for the unique substrate specificity of FoBGlcA. The amino acid residues involved in the catalytic activity of this enzyme were determined by evaluating the activity levels of various mutant enzymes based on the crystal structure analysis of the FoBGlcA reaction product complex. Database Atomic coordinates and structure factors (codes and ) have been deposited in the Protein Data Bank ().

Automated summary

In this study, a novel enzyme FoBGlcA with unique catalytic activity was isolated and characterized. The crystal structure of FoBGlcA revealed a high structural divergence compared to other GH79 beta-glucuronidases, and its unique substrate specificity may be attributed to distinct loop structure near the catalytic site. Evaluating the mutant enzymes based on the crystal structure of FoBGlcA reaction product complex determined the amino acid residues involved in the catalytic activity of the enzyme.