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

Thirumalaiselvi Ulaganathan, William Helbert, Moran Kopel, Ehud Banin, Miroslaw Cygler

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

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 languageEnglish
Pages (from-to)4026-4036
Number of pages11
JournalJournal of Biological Chemistry
Volume293
Issue number11
DOIs
StatePublished - 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.

FundersFunder 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

    Fingerprint

    Dive into the research topics of 'Structure-function analyses of a PL24 family ulvan lyase reveal key features and suggest its catalytic mechanism'. Together they form a unique fingerprint.

    Cite this