The crystal structure and insight into the substrate specificity of the α-L rhamnosidase RHA-P from Novosphingobium sp. PP1Y

Flavonoid natural products are well known for their beneficial antimicrobial, antitumor, and anti-inflammatory properties, however, some of these natural products often are rhamnosylated, which severely limits their bioavailability. The lack of endogenous rhamnosidases in the human GI tract not only prevents many of these glycosylated compounds from being of value in functional foods but also limits the modification of natural product libraries being tested for drug discovery. RHA-P is a catalytically efficient, thermostable alpha-L-rhamnosidase from the marine bacterium Novosphingobium sp. PP1Y that selectively hydrolyzes alpha-1,6 and alpha-1,2 glycosidic linkages between a terminal rhamnose and a flavonoid moiety. This work reports the 2.2 angstrom resolution crystal structure of RHA-P, which is an essential step forward in the characterization of RHA-P as a potential catalyst to increase the bioavailability of rhamnosylated natural compounds. The structure shows highly conserved rhamnose- and calcium-binding residues in a shallow active site that is housed in the (beta/alpha)(8) domain. In comparison to BT0986 (pdbID: 5MQN), the only known structure of an RHA-P homolog, the morphology, electrostatic potentials and amino acid composition of the substrate binding pocket are significantly different, offering insight into the substrate preference of RHA-P for glycosylated aryl compounds such as hesperidin, naringin, rutin, and quercitrin, over polysaccharides, which are preferred by BT0986. These preferences were further explored by using in silico docking, the results of which are consistent with the known kinetic data for RHA-P acting on different rhamnosylated flavonoids. Due to its promiscuity, relative thermostability compared to other known rhamnosidases, and catalytic efficiency even in significant concentrations of organic solvents, RHA-P continues to show potential for biocatalytic applications.