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

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.

A consensus-guided approach yields a heat-stable alkane-producing enzyme and identifies residues promoting thermostability

“…8. It has been shown in previous studies that the ligand approaches the diiron cluster from opposite side of the His-148 residue with its headgroup binding directly to the Fe2, which is the preferred iron for substrate binding (21,35). We also observed similar interactions in the case of the Np-ADO at 300 K where ligand appeared to be bound to the Fe2 (Fig.…”

“…The role of cysteine residues has been investigated in ADO from Nostoc punctiforme PCC 73102, and it was shown that Cys-71 is involved in maintaining the activity, structure, and stability of ADO (39). Certain amino acid residues present in the vicinity of the substrate channel were identified for successfully changing the substrate chain length selectivity of ADO (21). The ADO belongs to the nonheme dinuclear iron oxygenase family of enzymes that includes methane monooxygenase, type I ribonucleotide reductase, and ferritin (3, 35, 40 -42).…”

Directed evolution methods for overcoming trade‐offs between protein activity and stability

“…The importance of rationally designing enzyme libraries with amino acid diversity in specific regions has also been recently demonstrated in a variety of examples that involve optimization of substrate access tunnels of enzyme active sites to improve activity and stability . Several computational tools have been generated for identifying and engineering substrate access tunnels for diverse applications in enzyme engineering, including altering substrate selectivity (see references within a recent review).…”

Comparison of orthologous cyanobacterial aldehyde deformylating oxygenases in the production of volatile C3-C7 alkanes in engineered E. coli

“…The reaction is dependent on an external reducing system to provide altogether four electrons for the (i) activation of the di-iron center for oxygen binding, (ii) hemiacetal radical formation, and (iii) generation of the product complex ( Paul et al, 2013 ; Rajakovich et al, 2015 ). Characteristically, ADO has relatively broad substrate specificity towards different aldehydes, with the highest activity towards the native linear C16 and C18 precursors ( Khara et al, 2013; Bao et al, 2016 ; Zhang et al, 2016 ). This feature is accompanied by a low apparent catalytic turnover rate with reported k cat values below 0.02 s −1 in different contexts in vitro and in vivo ( Khara et al, 2013; Andre et al, 2013; Bao et al, 2016 ).…”

“…Characteristically, ADO has relatively broad substrate specificity towards different aldehydes, with the highest activity towards the native linear C16 and C18 precursors ( Khara et al, 2013; Bao et al, 2016 ; Zhang et al, 2016 ). This feature is accompanied by a low apparent catalytic turnover rate with reported k cat values below 0.02 s −1 in different contexts in vitro and in vivo ( Khara et al, 2013; Andre et al, 2013; Bao et al, 2016 ). Kinetic characterization has also revealed that the affinity towards the aldehyde substrates is poor, especially for the shorter precursors, with K M values in the millimolar range ( Bao et al, 2016 ).…”