Correction: New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product
[This corrects the article DOI: 10.1371/journal.pone.0175846.].
New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product
In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig’s blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.
Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue.
The amino acid sequences of the NH2 terminus and internal peptide fragments of a Rhodococcus rhodochrous J1 nitrilase were determined to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 750-base DNA fragment thus amplified was used as the probe to clone a 5.4-kilobase PstI fragment coding for the whole nitrilase. The nitrilase gene modified in the sequence upstream from the presumed ATG start codon was expressed to approximately 50% of the total soluble protein in Escherichia coli. The predicted amino acid sequence of the nitrilase gene showed similarity to that of the bromoxynil nitrilase from Klebsiella ozaenae. The 5,5'-dithiobis(2-nitrobenzoic acid) modification of the nitrilase from R. rhodochrous J1 resulted in inactivation with the loss of one sulfhydryl group/enzyme subunit. Of 4 cysteine residues in the Rhodococcus nitrilase, only Cys-165 is conserved in the Klebsiella nitrilase. Mutant enzymes containing Ala or Ser instead of Cys-165 did not exhibit nitrilase activity. These findings suggest that Cys-165 plays an essential role in the function of the active site.