The vanillyl alcohol oxidase/p-cresol methylhydroxylase (VAO/PCMH) flavoprotein family comprises a broad spectrum of enzymes capable of catalyzing the oxidative bioconversions of various substrates. Among them, pinoresinol hydroxylase (PinH) from the 4-alkylphenol oxidizing subgroup initiates the oxidative degradation of (+)-pinoresinol, a lignan important for both lignin structure and plant defense. In this study, we present a detailed biochemical and structural characterization of PinH from Pseudomonas sp., with focus on its substrate specificity and product formation. PinH was expressed in E. coli and purified as FAD-containing, soluble protein. The flavoenzyme catalyzes the hydroxylation of both (+)-pinoresinol and eugenol. Structural analysis reveals its dimeric form, non-covalent flavin binding, and a large active site. AlphaFold models of the PinH-cytochrome complex demonstrate cytochrome's dual role in electron transfer and modulating PinH's conformation. A distinctive feature of PinH is a large cavity that hosts its multi-ring (+)-pinoresinol substrate. The capability of converting bulky lignans is particularly attractive for biotechnological applications aimed at producing high-value compounds from phenolic precursors. These insights expand our knowledge on the structure and mechanism of the VAO/PCMH flavoenzyme family members.

Biochemical and structural insights into pinoresinol hydroxylase from Pseudomonas sp

Guerriere, Teresa Benedetta
Methodology
;
Fraaije, Marco W
Supervision
;
Mattevi, Andrea
Supervision
2025-01-01

Abstract

The vanillyl alcohol oxidase/p-cresol methylhydroxylase (VAO/PCMH) flavoprotein family comprises a broad spectrum of enzymes capable of catalyzing the oxidative bioconversions of various substrates. Among them, pinoresinol hydroxylase (PinH) from the 4-alkylphenol oxidizing subgroup initiates the oxidative degradation of (+)-pinoresinol, a lignan important for both lignin structure and plant defense. In this study, we present a detailed biochemical and structural characterization of PinH from Pseudomonas sp., with focus on its substrate specificity and product formation. PinH was expressed in E. coli and purified as FAD-containing, soluble protein. The flavoenzyme catalyzes the hydroxylation of both (+)-pinoresinol and eugenol. Structural analysis reveals its dimeric form, non-covalent flavin binding, and a large active site. AlphaFold models of the PinH-cytochrome complex demonstrate cytochrome's dual role in electron transfer and modulating PinH's conformation. A distinctive feature of PinH is a large cavity that hosts its multi-ring (+)-pinoresinol substrate. The capability of converting bulky lignans is particularly attractive for biotechnological applications aimed at producing high-value compounds from phenolic precursors. These insights expand our knowledge on the structure and mechanism of the VAO/PCMH flavoenzyme family members.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/1519095
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? 0
social impact