{"doi":"10.1016/j.jbc.2024.105647","title":"Dirigent isoflavene-forming PsPTS2: 3D structure, stereochemical, and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea","abstract":"Pea phytoalexins (–)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2ʹ-dihydroxy-4ʹ,5ʹ-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity towards four possible 7,2ʹ-dihydroxy-4ʹ,5ʹ-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis -(3 R, 4 R )-DMDI into DMDIF 20-fold faster than the trans -(3 R ,4 S )-isomer. The 4 R -configured substrate's near β-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4 S -isomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4 R -configured β-axial OH was closest to Asp 51 , whereas 4 S -isomer's α-equatorial OH was further away. Neither cis -(3 S ,4 S )- nor trans -(3 S ,4 R )-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis -(3 R, 4 R )-7,2′-dihydroxy-4′-methoxyisoflavan-4-ol [ cis -(3 R, 4 R )-DMI] and C3/C4 stereoisomers to give 2ʹ,7-dihydroxy-4ʹ-methoxyisoflav-3-ene (DMIF). DP homologs may exist in licorice ( Glycyrrhiza pallidiflora ) and tree legume Bolusanthus speciosus, as DMIF occurs in both species. PsPTS1 utilized cis -(3 R, 4 R )-DMDI to give (–)-maackiain 2200-fold more efficiently than with cis -(3 R ,4 R )-DMI. PsPTS1 also slowly converted trans -(3 S ,4 R )-DMDI into (+)-maackiain, reflecting the better 4 R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (–)-maackiain, via identical DP-engendered mono-QM bound intermediate generation, which PsPTS2 either re-aromatizes to give DMDIF or PsPTS1 intramolecularly cyclizes to afford (–)-maackiain. Substrate docking simulations using PsPTS2 and PsPTS1 indicate cis -(3 R, 4 R )-DMDI binds in the anti -configuration in PsPTS2 to afford DMDIF, and the syn -configuration in PsPTS1 to give maackiain.","journal":"Journal of Biological Chemistry","year":2024,"id":457046,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.946,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1076046,"name":"Syed Moinuddin","orcid":"0000-0002-9907-416X","position":1,"is_corresponding":false},{"id":1076047,"name":"Rhodesia M. 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