Expression, purification, and antibody purification for the enzyme methylthiooxobutyrate reductase in algal dimethylsulfoniopropionate biosynthesis

Abstract

Many marine algae produce the organosulfur compound dimethylsulfoniopropionate (DMSP). This compound is thought to be an important osmoprotectant to both producers and grazers. It is known to counteract stressors algae encounter in the euphotic zone such as fluctuating salinity, ultraviolet (UV) irradiation, and grazing. When liberated into marine surface waters, dissolved DMSP becomes an important source of carbon and reduced sulfur for the marine microbial community and through its breakdown into dimethylsulfide (DMS), the chief biogenic vehicle for sulfur transit into the atmosphere. The importance of DMSP has been well studied and known for decades, but less is known about the enzymes that govern its biosynthesis in marine algae. Earlier studies identified and partially characterized the 4 biosynthetic enzymes from tissue extracts of the macroalgae Ulva intestinalis but lacked thorough analysis of the individual enzymes. The sequencing of the Ulva mutabilis genome revealed a megasynthetase encoding a tripartite fusion protein with domains consistent with catalyzing the last three enzymatic steps of DMSP biosynthesis. This discovery enabled the design of recombinant proteins of the individual domains with encoded affinity tags for chromatographic purification to enable the isolation of pure enzyme. This project sought to leverage these recombinant variants to expand the understanding of the second-step enzyme, 4-methylthio-2-oxobutyrate (MTOB) reductase, the putative 2-D-hydroxyacid dehydrogenase enzyme that catalyzes the NADPH-coupled stereospecific reduction of MTOB to D-4-methylthio-2-hydroxybutyrate (MTHB). This study finds that recombinant C-terminally hexahistidine-tagged MTOB reductase is likely inactive, that purification should be further refined to eliminate contaminating proteins, and that MTOB reductase-specific antibody purification for this protein was unsuccessful. Additionally, this project has produced 2 new recombinant versions of MTOB reductase for future testing.