Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1

Sirirungruang, Sasilada; Blay, Vincent; Scott, Yasmine F.; Pereira, Jose H.; Hammel, Michal; Barnum, Collin R.; Adams, Paul D.; Shih, Patrick M.

doi:10.1016/j.jbc.2024.107602

Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1

Journal Article · Tue Aug 20 00:00:00 EDT 2024 · Journal of Biological Chemistry

DOI:https://doi.org/10.1016/j.jbc.2024.107602· OSTI ID:2406503

^[1]; ^[2]; Scott, Yasmine F. ^[3]; Pereira, Jose H. ^[4]; Hammel, Michal ^[5]; Barnum, Collin R. ^[6]; ^[4]; Shih, Patrick M. ^[7]

Univ. of California, Berkeley, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Suranaree University of Technology, Nakhon Ratchasima (Thailand)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)
Univ. of California, Berkeley, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Univ. of California, Davis, CA (United States)
Univ. of California, Berkeley, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

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Research Organization:: Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS); National Institutes of Health (NIH)

Sponsoring Organization:: USDOE; USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2406503

Alternate ID(s):: OSTI ID: 2480479

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 9 Vol. 300; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1

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