Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 4026-4036 |
| Number of pages | 11 |
| Journal | Journal of Biological Chemistry |
| Volume | 293 |
| Issue number | 11 |
| DOIs | |
| State | Published - 16 Mar 2018 |
Bibliographical note
Publisher Copyright:© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Funding
This work was supported by Grant 155375-2012-RGPIN from the Natural Sci-ence and Engineering Council (NSERC) (to M. C.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1–S3. The atomic coordinates and structure factors (codes 6BYP, 6BYX, and 6BYT) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1 To whom correspondence should be addressed: Dept. of Biochemistry, Uni-versity of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada. Tel.: 306-966-4361; E-mail: [email protected]. Acknowledgments—We thank Drs. Jeremy Lee and David R. Palmer for helpful discussions. We acknowledge the Protein Characterization and Crystallization Facility, College of Medicine, University of Saskatchewan, for access to the crystallization robot. Research described in this paper was performed using beamline 08ID-1 at the Canadian Light Source, which is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the Canadian Institutes of Health Research.
| Funders | Funder number |
|---|---|
| Government of Saskatchewan | |
| Natural Sci-ence and Engineering Council | |
| University of Saskatchewan | |
| Canadian Institutes of Health Research | |
| Natural Sciences and Engineering Research Council of Canada | |
| Western Economic Diversification Canada | |
| National Research Council Canada | |
| Canada Foundation for Innovation |
