학술논문
Dirigent isoflavene-forming PsPTS2: 3D structure, stereochemical, and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea.
Document Type
Article
Author
Qingyan Meng; Moinuddin, Syed G. A.; Celoy, Rhodesia M.; Smith, Clyde A.; Young, Robert P.; Costa, Michael A.; Freeman, Rachel A.; Masashi Fukaya; Doo Nam Kim; Cort, John R.; Hawes, Martha C.; van Etten, Hans D.; Pandey, Pankaj; Chittiboyina, Amar G.; Ferreira, Daneel; Davin, Laurence B.; Lewis, Norman G.
Source
Subject
*MOLECULAR orbitals
*STEREOISOMERS
*CIRCULAR dichroism
*NUCLEAR magnetic resonance spectroscopy
*STEREOCHEMISTRY
*PEAS
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Language
ISSN
0021-9258
Abstract
Pea phytoalexins (-)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2'- dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity toward four possible 7,2'-dihydroxy-4',5'- 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- (3R,4R)-DMDI into DMDIF 20-fold faster than the trans- (3R,4S)-isomer. The 4R-configured substrate's near β-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4Sisomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured β-axial OH was closest to Asp51, whereas 4S-isomer's α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis-(3R,4R)-7,20-dihydroxy- 4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and C3/C4 stereoisomers to give 2',7-dihydroxy-4'-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-(3R,4R)-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3R,4R)- DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (-)-maackiain, via identical DPengendered 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-(3R,4R)-DMDI binds in the anti-configuration in PsPTS2 to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain. [ABSTRACT FROM AUTHOR]