Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length.

Xuefeng Lu,Luyao Bao,Jian-Jun Li,Chenjun Jia,Mei Li

doi:10.1186/s13068-016-0596-9

Abstract

BackgroundAldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C4 to C18 aldehydes, which is not suitable for producing fuels with different properties or different chain lengths.ResultsBased on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C14,16,18 aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C4,6,7 aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C6–10 aldehydes and L198F for C7–10 aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane.ConclusionsSome amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0596-9) contains supplementary material, which is available to authorized users.

Highlights

Aldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic path‐ way of fatty alk(a/e)nes in cyanobacteria
Andre et al reported that cADO was reversibly inhibited by H2O2 originating from poor coupling of reductant consumption with alk(a/e)ne formation, and the kinetics of cADO towards aldehyde substrates of carbon chain lengths between 8 and 18 carbons showed that cADO did not exhibit strong chain-length specificity with respect to its substrates [15]. cADO produces n-1 aldehydes and alcohols in addition to alk(a/e)ne [16]
Identification of the amino acids that may influence the substrate specificity of cADO According to the crystal structure of cADO-1593 from Synechococcus elongatus strain PCC7942 (PDB code: 4RC5) [17], amino acids involved in the substrate channel were identified, including Tyr21, Ile24, Ile27, Val28, Gly31, Phe67, Cys70, Phe86, Phe87, Phe117, Ala118, A121, Tyr122, Tyr125, Val184, Met193 and Leu198 (Fig. 1)

Summary

Introduction

Aldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic path‐ way of fatty alk(a/e)nes in cyanobacteria. ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C4 to C18 aldehydes, which is not suitable for producing fuels with different properties or different chain lengths. It has been accepted that one of the enzymatic pathways producing alk(a/e)nes is derived from fatty acyl-ACP or -CoA in a two-step reaction: fatty acyl-ACP or -CoA is first reduced into fatty aldehyde by acyl-ACP or -CoA reductase, fatty aldehyde is converted into alk(a/e) ne by aldehyde decarbonylase ( renamed as aldehyde-deformylating oxygenase, ADO). In 2013, Akhtar et al reported that a carboxylic acid reductase (CAR) from Mycobacterium marinum could convert a wide range of aliphatic fatty acids (C6–C18) into corresponding aldehydes, which can be transformed into fatty alkane by ADO [6]. Mechanistic studies have demonstrated that a radical intermediate is involved in the cADO-catalyzed reaction, and a possible

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