Sphingobacterium sp. T2 Manganese Superoxide Dismutase Catalyzes the Oxidative Demethylation of Polymeric Lignin via Generation of Hydroxyl Radical.

Vilmos Fülöp,Stéphanie Baumberger,Timothy D H Bugg,Goran M M Rashid,Betty Cottyn,Rachael C Wilkinson,Xiaoyang Zhang

doi:10.1021/acschembio.8b00557

Abstract

Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl-Cα oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. 18O labeling studies on the SpMnSOD-catalyzed oxidation of lignin model compound guiaiacylglycerol-β-guaiacyl ether indicated that the an oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative 31P NMR spectroscopy demonstrated 20-40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of 4.1 molar equivalents of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to 31P NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn center for hydroxyl radical generation.

Highlights

The aromatic heteropolymer lignin is a major constituent (15-25%) of plant cell wall lignocellulose, and represents a possible raw material for generation of renewable aromatic chemicals by depolymersation
T2 manganese superoxide dismutase enzymes in lignin oxidation[10] is unusual, since manganese superoxide dismutase is normally involved in protection against oxidative stress, it was of particular interest to elucidate the catalytic mechanism for this reaction
In this paper we provide evidence that treatment of polymeric lignin with SpMnSOD1 causes increases in phenolic OH content, consistent with oxidative demethylation, a reaction observed with vanillic acid (3) and observed previously with guaiacol.[10]

Summary

Introduction

The aromatic heteropolymer lignin is a major constituent (15-25%) of plant cell wall lignocellulose, and represents a possible raw material for generation of renewable aromatic chemicals by depolymersation. Demethylase enzymes usually generate formaldehyde via oxidation of the methyl group C-H bond, hydroxyl radical has been shown to react with methoxy-substituted aromatic compounds via an ipso-reaction, with loss of methanol via C-O bond cleavage.[19] Attempts to detect formaldehyde using the colorimetric assay of Li et al[20] gave no observed signal from 1,4-dimethoxybenzene, and only 2-3 μM formaldehyde from 1 mM vanillic acid (see Supporting Information Figure S22).

Results

Conclusion