Structure-function analyses of a PL24 family ulvan lyase reveal key features and suggest its catalytic mechanism

Ulvan is a major cell wall component of green algae of the genus Ulva, and some marine bacteria encode enzymes that can degrade this polysaccharide. The first ulvan-degrading lyases have been recently characterized, and several putative ulvan lyases have been recombinantly expressed, confirmed as ulvan lyases, and partially characterized. Two families of ulvan-degrading lyases, PL24 and PL25, have recently been established. The PL24 lyase LOR-107 from the bacterial Alteromonadales sp. strainLORdegrades ulvan endolytically, cleaving the bond at the C4 of a glucuronic acid. However, the mechanism and LOR-107 structural features involved are unknown.Wepresent here the crystal structure of LOR-107, representing the first PL24 family structure. We found that LOR-107 adopts a sevenbladed β-propeller fold with a deep canyon on one side of the protein. Comparative sequence analysis revealed a cluster of conserved residues within this canyon, and site-directed mutagenesis disclosed several residues essential for catalysis. We also found that LOR-107 uses the His/Tyr catalytic mechanism, common to several PL families. We captured a tetrasaccharide substrate in the structures of two inactive mutants, which indicated a two-step binding event, with the first substrate interaction near the top of the canyon coordinated by Arg320, followed by sliding of the substrate into the canyon toward the active-site residues. Surprisingly, the LOR-107 structure was very similar to that of the PL25 family PLSV-3936, despite only∼14% sequence identity between the two enzymes. On the basis of our structural and mutational analyses, we propose a catalytic mechanism for LOR-107 that differs from the typical His/Tyr mechanism.