Cloning, expression, purification, and characterization of novel initiating enzyme in Pseudo-nitzschia pungens dimethylsulfoniopropionate biosynthesis pathway

Abstract

Dimethylsulfoniopropionate (DMSP) is a tertiary sulfonium compound synthesized by marine bacteria, algae, and some higher plants. This secondary metabolite functions as an osmolyte, a herbivore deterrent, an antioxidant, and a cryoprotectant. In algae, the biosynthesis of DMSP is catalyzed by a four-enzyme pathway, which begins with the conversion of methionine to methylthio-2-oxobutyrate. While it is known that Ulva macroalgae species requires a 2-oxoglutarate:methionine aminotransferase for the first step in the DMSP pathway, little research has been performed on other alga species. Genome searches by Dr. Waller identified an enzyme in diatom species of Pseudo-nitzschia, termed MetDA here, which based on strong genetic associations with known DMSP enzymes is believed to be a novel initiating enzyme for the DMSP pathway. Cloning the MetDA gene into three vectors was attempted. Two constructs of the recombinant MetDA protein were successfully expressed in E. coli cells and purified using affinity column chromatography. A 2,4-dinitrophenylhydrazine assay was developed to characterize the deamination activity of MetDA. A perhaps promising result shows MetDA acts as a methionine deaminase with a NAD+ prosthetic group with an activity rate of 18.3 pmol per min per μg protein, which is in a similar range of other DMSP enzymes. This newly discovered enzyme gives metabolic engineers another option for genetic engineering of plants or bacteria to enhance their osmotic and oxidative stress tolerance.