Protein engineering of the aldoxime dehydratase from Bacillus sp. OxB-1 based on a rational sequence alignment approach

Keiko Oike,Yasuhisa Asano,Harald Gröger,Daisuke Matsui,Jens Sproß

doi:10.1038/s41598-021-92749-0

Keiko Oike, Yasuhisa Asano + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-92749-0

Copy DOI

Abstract

Recently, the program INTMSAlign_HiSol for identifying aggregation hotspots in proteins only requiring secondary structure data was introduced. We explored the utility of this program further and applied it for engineering of the aldoxime dehydratase from Bacillus sp. OxB-1. Towards this end, the effect of inverting the hydropathy at selected positions of the amino acid sequence on the enzymatic activity was studied leading to 60% of our constructed variants, which showed improved activity. In part, this activity increase can be rationalised by an improved heme incorporation of the variants. For example, a single mutation gave a 1.8 fold increased enzymatic activity and 30% improved absolute heme incorporation.

Highlights

The program INTMSAlign_HiSol for identifying aggregation hotspots in proteins only requiring secondary structure data was introduced
OxB-1 (OxdB), which was isolated from soil[1]. This enzyme naturally catalyses the dehydration of E- and Z-phenylacetaldoxime (E- and Z-PAOx) to phenylacetonitrile (PAN). It was purified as wild-type enzyme and expressed heterologously in Escherichia coli (E. coli)[5,6]
YH3-3 was targeted by directed evolution via random mutagenesis in order to improve the catalytic efficiency for the dehydration of 2-furfurylaldoxime to 2-furfuryl nitrile, a biorenewable feedstock-based nitrile with several applications in the chemical industry[29]

Summary

Introduction

The program INTMSAlign_HiSol for identifying aggregation hotspots in proteins only requiring secondary structure data was introduced. Towards this end, the effect of inverting the hydropathy at selected positions of the amino acid sequence on the enzymatic activity was studied leading to 60% of our constructed variants, which showed improved activity. Aldoxime dehydratases (Oxds; E.C. 4.99) are special heme containing lyases involved in the “aldoxime–nitrilepathway” of microbes (Fig. 1). These enzymes catalyse the dehydration of aldoximes to nitriles, which are metabolised to carboxylic acids by nitrile hydratases and amidases or by n itrilases[1,2,3,4]. OxB-1 (OxdB), which was isolated from soil[1] This enzyme naturally catalyses the dehydration of E- and Z-phenylacetaldoxime (E- and Z-PAOx) to phenylacetonitrile (PAN). N-771 (OxdRE)[33] and Pseudomonas chloraphis B23 (OxdA)[34] are the only ones known up to now

Methods

Results

Discussion

Conclusion