Comparison of Biochemical Properties of the Original and Newly Identified Oleate Hydratases from Stenotrophomonas maltophilia

Abstract

Oleate hydratases (OhyAs) catalyze the conversion of unsaturated fatty acids to 10-hydroxy fatty acids, which are used as precursors of important industrial compounds, including lactones and -hydroxycarboxylic and ␣, -dicarboxylic acids. The genes encoding OhyA and a putative fatty acid hydratase in Stenotrophomonas maltophilia were identified by genomic analysis. The putative fatty acid hydratase was purified and identified as an oleate hydratase (OhyA2) based on its substrate specificity. The activity of OhyA2 as a holoenzyme was not affected by adding cofactors, whereas the activity of the original oleate hydratase (OhyA1) showed an increase. Thus, all characterized OhyAs were categorized as either OhyA1 or OhyA2 based on the activities of holoenzymes upon adding cofactors, which were determined by the type of the fourth conserved amino acid of flavin adenine dinucleotide (FAD)-binding motif. The hydration activities of S. maltophilia OhyA2 toward unsaturated fatty acids, including oleic acid, palmitoleic acid, linoleic acid, ␣-linolenic acid, and ␥-linolenic acid, were greater than those of OhyA1. Moreover, the specific activity of S. maltophilia OhyA2 toward unsaturated fatty acids, with the exception of ␥-linolenic acid, was the highest among all reported OhyAs. IMPORTANCE All characterized OhyAs were categorized as OhyA1s or OhyA2s based on the different properties of the reported and newly identified holo-OhyAs in S. maltophilia upon the addition of cofactors. OhyA2s showed higher activities toward polyunsaturated fatty acids (PUFAs), including linoleic acid, ␣-linolenic acid, and ␥-linolenic acid, than those of OhyA1s. This suggests that OhyA2s can be used more effectively to convert plant oils to 10-hydroxy fatty acids because plant oils contain not only oleic acid but also PUFAs. The hydration activity of the newly identified OhyA2 from S. maltophilia toward oleic acid was the highest among the activity levels reported so far. Therefore, this enzyme is an efficient biocatalyst for the conversion of plant oils to 10-hydroxy fatty acids, which can be further converted to important industrial materials. KEYWORDS Stenotrophomonas maltophilia, enzyme characterization, oleate hydrataseO leate hydratase (OhyA) converts unsaturated fatty acids to 10-hydroxy fatty acids, which are then converted to ␥-lactones by the yeasts Candida boidinii and Waltomyces lipofer (1, 2). ␥-Lactones are flavor compounds used in food and cosmetics (3). Multistep enzymatic reactions involving OhyA, alcohol dehydrogenase, Baeyer-Villiger monooxygenases, and esterase convert unsaturated fatty acids to -hydroxycarboxylic and ␣, -dicarboxylic acids (4), which are used in the preparation of resins, nylons, plastics, and lubricants (5). An effective OhyA is necessary for the efficient production of ␥-lactones, -hydroxycarboxylic acids, and ␣, -dicarboxylic acids.

On the current role of hydratases in biocatalysis

“…All currently known FAHs share a conserved N-terminal nucleotide binding motif for non-covalent attachment of the essential flavin adenine dinucleotide (FAD) cofactor despite their high overall sequence diversity (Wierenga et al 1986 ; Kleiger and Eisenberg 2002 ). Therefore, all FAHs characterised so far are flavin-dependent proteins (Volkov et al 2010 ; Joo et al 2012a ; Engleder et al 2015 ; Hirata et al 2015 ; Kang et al 2017 ). Since the redox (reduction/oxidation) state of FAD does not change during substrate conversion, FAHs are belonging to the approx.…”

“…The most probable role of FAD in FAHs comprises the correct assembly of amino acids in the active site (Engleder et al 2015 ). Additionally, a beneficial effect of FAD reduction to its two-electron reduced state was observed for OA hydration by OhyA, as well as the linoleic acid (LA) Δ9 hydratase from Lactobacillus plantarum AKU 1009a (CLA-HY) and OhyA1, but not for OhyA2 from Stenotrophomonas maltophilia (Takeuchi et al 2014 ; Engleder et al 2015 ; Kang et al 2017 ). This is possibly due to facilitating protonation of the carbon-carbon double bond of the substrates (Macheroux et al 2005 ; Engleder et al 2015 ).…”

Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicum NRRL 5767 by CRISPR/Cas9 genome editing technology

“…The FAD plays a dual role in the proper organization of the active site and the stabilization of the partial positive charge in the putative transition state of the hydration reaction [44]. Although many Ohases require FAD for activity [33,35,36,49], some Ohases do not depend on FAD [45,50], including the two Ohase isozymes (NcOhy1 and NcOhy2) from this study.…”

Emerging roles of metabolites of ω3 and ω6 essential fatty acids in the control of intestinal inflammation

“…pyogenes (77), Stenotrophomonas nitritireducens (78) and Steno. maltophilia (79). In addition to CLA-HY in L. plantarum , enzymes that generate HYA from linoleic acid include FA-HY2 in L. acidophilus (80), SPH in Strep.…”

“…pyogenes (77) and OhyA1-2 in Steno. maltophilia (79); all of these were annotated previously as myosin-cross-reactive antigen (MCRA) proteins (Fig. 4).…”