Identification of residues important for the activity of aldehyde-deformylating oxygenase through investigation into the structure-activity relationship

Abstract

Background: Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, cADO (cyanobacterial ADO) showed extreme low activity with the k _cat value below 1 min^-1, which would limit its application in biofuel production. To identify the activity related key residues of cADO is urgently required.

Results: The amino acid residues which might affect cADO activity were identified based on the crystal structures and sequence alignment of cADOs, including the residues close to the di-iron center (Tyr39, Arg62, Gln110, Tyr122, Asp143 of cADO-1593), the protein surface (Trp 178 of cADO-1593), and those involved in two important hydrogen bonds (Gln49, Asn123 of cADO-1593, and Asp49, Asn123 of cADO-sll0208) and in the oligopeptide whose conformation changed in the absence of the di-iron center (Leu146, Asn149, Phe150 of cADO-1593, and Thr146, Leu148, Tyr150 of cADO-sll0208). The variants of cADO-1593 from Synechococcus elongatus PCC7942 and cADO-sll0208 from Synechocystis sp. PCC6803 were constructed, overexpressed, purified and kinetically characterized. The k _cat values of L146T, Q49H/N123H/F150Y and W178R of cADO-1593 and L148R of cADO-sll0208 were increased by more than two-fold, whereas that of R62A dropped by 91.1%. N123H, Y39F and D143A of cADO-1593, and Y150F of cADO-sll0208 reduced activities by ≤ 20%.

Conclusions: Some important amino acids, which exerted some effects on cADO activity, were identified. Several enzyme variants exhibited greatly reduced activity, while the k _cat values of several mutants are more than two-fold higher than the wild type. This study presents the report on the relationship between amino acid residues and enzyme activity of cADOs, and the information will provide a guide for enhancement of cADO activity through protein engineering.

Keywords: Aldehyde-deformylating oxygenase; Fatty alk(a/e)ne; Site-directed mutagenesis; Structure-activity relationship; Synechococcus elongatus PCC7942; Synechocystis sp. PCC6803.

Fig. 1

cADO-catalyzed reaction [–16]

Fig. 2

Identified residues based on the crystal structure of ADO from Synechococcuselongatus PCC7942 (1593; PDB code:4RC5). The identified residues include those close to the di-iron center (Tyr39, Gln110, Tyr122), the protein surface (Trp178), and involved in the hydrogen-bonding network (Arg62, Asp143) and the oligopeptide whose conformation changed (Leu/Thr146, Leu148, Asn149 and Tyr/Phe150) in the absence of the diiron center

Fig. 3

Sequence alignment of 1593, sll0208 and PMT1231. The residues investigated in this paper are labelled with black dots above the sequence

Fig. 4

Two hydrogen bonds in sll0208. The hydrogen-bond lengths between Asn123 and Tyr145 and between Tyr150 and Asp49 are 3.0 and 2.4 Å respectively

Fig. 5

Structural superimposition of L194A of PMT1231 (palecyan, PDB code: 4PGI) and 1593 (light pink, PDB code: 4QUW). Arg191 of PMT1231, Trp178 of 1593 and two substrate-binding modes were shown