Lignin, a complex aromatic polymer, represents a significant obstacle in lignocellulosic biomass utilization. The polymerization of lignin occurs by radical couplings, which mainly form ether and C-C bonds between monolignol units. The chemical stability of these bonds between monolignol units causes the recalcitrant nature of lignin. Since the Cα-Cβ double bond in the monolignols is a crucial chemical feature for the radical coupling, reduction of the double bond would decrease the degree of lignin polymerization, avoiding the recalcitrance of lignin. To develop a method of lignin engineering, we have focused on alkenal double bond reductases (DBR), which can reduce the Cα-Cβ double bond of a monolignol precursor. Here, a novel bacterial DBR from Parvibaculum lavamentivorans DS-1 (PlDBR) was found. This enzyme can reduce the side-chain double bond of coniferaldehyde (CALD) and has a 41% amino-acid sequence identity with CALD DBR from Arabidopsis thaliana (AtDBR). The crystal structure of the PlDBR showed that it has a larger substrate-binding pocket than AtDBR, conferring broader substrate specificity on the former. Structural and mutation analyses of PlDBR and AtDBR suggested that Tyr51 and Try252 are critical residues for the catalytic activity of PlDBR. In addition, Tyr81 of AtDBR appears to cause substrate inhibition. Replacing Tyr81 of AtDBR with a smaller amino-acid residue, as in the AtDBR variants Tyr81Leu and Tyr81Ala, resulted in a substantially higher CALD-reducing activity compared to the wild type. These variants would be promising candidates for lignin manipulation to decrease the recalcitrance of lignocellulosic biomass.
Keywords: Alkenal double bond reductase; Coniferaldehyde; Crystal structure; Enzyme engineering; Lignin manipulation.
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