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Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6
Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alco...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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American Society for Biochemistry and Molecular Biology
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191317/ https://www.ncbi.nlm.nih.gov/pubmed/33965373 http://dx.doi.org/10.1016/j.jbc.2021.100758 |
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author | Kuatsjah, Eugene Chan, Anson C.K. Katahira, Rui Haugen, Stefan J. Beckham, Gregg T. Murphy, Michael E.P. Eltis, Lindsay D. |
author_facet | Kuatsjah, Eugene Chan, Anson C.K. Katahira, Rui Haugen, Stefan J. Beckham, Gregg T. Murphy, Michael E.P. Eltis, Lindsay D. |
author_sort | Kuatsjah, Eugene |
collection | PubMed |
description | Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from β-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼10(6) M(−1) s(−1)) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity. |
format | Online Article Text |
id | pubmed-8191317 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-81913172021-06-16 Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6 Kuatsjah, Eugene Chan, Anson C.K. Katahira, Rui Haugen, Stefan J. Beckham, Gregg T. Murphy, Michael E.P. Eltis, Lindsay D. J Biol Chem Research Article Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from β-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼10(6) M(−1) s(−1)) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity. American Society for Biochemistry and Molecular Biology 2021-05-07 /pmc/articles/PMC8191317/ /pubmed/33965373 http://dx.doi.org/10.1016/j.jbc.2021.100758 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Kuatsjah, Eugene Chan, Anson C.K. Katahira, Rui Haugen, Stefan J. Beckham, Gregg T. Murphy, Michael E.P. Eltis, Lindsay D. Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6 |
title | Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6 |
title_full | Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6 |
title_fullStr | Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6 |
title_full_unstemmed | Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6 |
title_short | Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6 |
title_sort | structural and functional analysis of lignostilbene dioxygenases from sphingobium sp. syk-6 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191317/ https://www.ncbi.nlm.nih.gov/pubmed/33965373 http://dx.doi.org/10.1016/j.jbc.2021.100758 |
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