Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase

Abstract

Stilbenes are diphenyl ethene compounds produced naturally in a wide variety of plant species and some bacteria. Stilbenes are also derived from lignin during kraft pulping. Stilbene cleavage oxygenases (SCOs) cleave the central double bond of stilbenes, forming two phenolic aldehydes. Here, we report the structure of an SCO. The X-ray structure of NOV1 from Novosphingobium aromaticivorans was determined in complex with its substrate resveratrol (1.89 Å), its product vanillin (1.75 Å), and without any bound ligand (1.61 Å). The enzyme is a seven-bladed β-propeller with an iron cofactor coordinated by four histidines. In all three structures, dioxygen is observed bound to the iron in a side-on fashion. These structures, along with EPR analysis, allow us to propose a mechanism in which a ferric-superoxide reacts with substrate activated by deprotonation of a phenol group at position 4 of the substrate, which allows movement of electron density toward the central double bond and thus facilitates reaction with the ferric superoxide electrophile. Correspondingly, NOV1 cleaves a wide range of other stilbene-like compounds with a 4′-OH group, offering potential in processing some solubilized fragments of lignin into monomer aromatic compounds.tilbenes are diphenyl ethene compounds that are produced naturally in a wide variety of plant species and some bacteria. One stilbene derivative of note is resveratrol, which is a plant phytoalexin abundant in grapes and peanuts. Studies have demonstrated numerous health benefits related to the consumption of resveratrol, which is correlated with reduced cardiovascular disease and cancer (1). Lignostilbene α,β-dioxygenase (LSD, EC 1.13.11.43), originally observed in Sphingomonas paucimobilis, was the first enzyme shown to cleave the central double bond of stilbenes, forming two phenolic aldehydes (2, 3). Subsequently, NOV1 and NOV2 (4) from Novosphingobium aromaticivorans, Rco1 (5) from Ustilago maydis, and CAO-1 (6) from Neurospora crassa were also shown to be stilbene cleaving oxygenases (SCOs). SCOs are related to carotenoid cleavage oxygenases (CCOs), which are enzymes that oxidatively cleave β-carotene or apocarotenoids. Carotenoids are a diverse class of molecules that play important roles in photosynthesis, immune function, and light perception in the eye. CCOs have been studied in great detail, including several crystal structures (7-9).Here, we present the X-ray structure of an SCO, NOV1 from N. aromaticivorans (NOV1). The structure was determined in complex with a representative substrate (resveratrol), a representative product (vanillin), and without ligand bound. We have also observed the ternary complex with oxygen and substrate or product bound, which has not been previously detected in a crystal structure of any CCO-related enzyme. Despite being related to CCOs, this structure of NOV1 shows several key differences that are indicative of their disparate substrate specificities. Moreover, the observed placement of Fe, O 2 , and the phenolic substrate resveratrol in th...

Structure and Spectroscopy of Alkene-Cleaving Dioxygenases Containing an Atypically Coordinated Non-Heme Iron Center

“…58 Interestingly, a dimeric assembly like that of CAO1 is also present in the NOV1 crystal structure, suggesting a conserved interface. 24 …”

“…Similar findings were reported for NOV1, and the formation of an Fe-NO complex in the absence of substrate was taken as evidence of the similar reactivity with O 2 . 24 However, such a conclusion is problematic because of the higher Fe(II) binding affinity of NO relative to O 2 . The literature provides several examples of non-heme iron oxygenases with known sequential binding of substrate and O 2 that are nevertheless reactive toward NO without substrate.…”

Mechanistic Insights into a Stibene Cleavage Oxygenase NOV1 from Quantum Mechanical/Molecular Mechanical Calculations

“…So far, four enzymes, NOV1 and NOV2 from Novosphingobium aromaticivorans , Rco1 from Ustilago maydis , and CAO1 from Neurosporacrassa were identified as resveratrol cleavage enzymes that catalyze the oxidative cleavage of resveratrol at the interphenyl double bond, forming two phenolic aldehydes. Together with lignostilbene α,β‐dioxygenase (LSD) from Sphingomonas paucimobilis , these enzymes are recently classified as the members of stibene cleavage oxygenases (SCOs), which catalyze the double bond cleavage of stilbenes . SCOs are related to carotenoid cleavage oxygenases (CCOs), which are non‐heme Fe(II)‐dependent enzymes that catalyze oxidative cleavage of double bonds in the conjugated carbon chain of carotenoids by dioxygen.…”

“…The geometric and electronic structures of the Fe−O 2 complex have attracted great interest in the non‐heme iron enzymes and their biomimetic compounds since it is the first oxygen species during the dioxygen activation and could act as an active oxidant. Recently, a breakthrough for resveratrol cleavage enzymes has been made on the crystallographic structure of NOV1 . Like CCOs, the non‐heme iron center of NOV1 is coordinated by four histidine ligands.…”

Reply to Kiser: Dioxygen binding in NOV1 crystal structures

“…As might be expected, the electron density ranges from highly symmetric to highly irregular. For example, naphthalene 1,2-dioxygenase [PDB ID code 1O7N (2)], cited by Kiser as a comparable enzyme, has a dioxygen bound in a sideon manner to an iron in almost exactly the same orientation as the NOV1 structures with and without substrate (3). Like NOV1, the density surrounding the dioxygen is not completely symmetric, and there are many more examples in the PDB.…”