Molecular Cloning, Expression And Biological Function Of Salsolinol Synthase

Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder. Therehave been increasing evidences indicating the endogenous dopamine-derived catecholisoquinolines whose structure is similar with MPTP, may be a possible candidate ofdopaminergic neurotoxins to elicit PD. Salsolinol (6,7-dihydroxy-1,2,3,4-tetrahydro-isoquinoline, Sal) as one kind of endogenous catechol isoquinoline, it can be detected inhuman brain and urine. Salsolinol synthase is one of the most important enzymes involvedin the formation of catechol isoquinoline neurotoxin. It is a novel enzyme which cancondensate dopamine and acetaldehyde to salsolinol, and causes the death of dopaminergicneurons. It is the first key enzyme in the metabolic pathway of catechol isoquinolineswhich is closely related to the pathogenesis of PD, and it directly affects the synthesis ofSal and its derivative metabolism in vivo.Based on our previous studies the existence of Sal synthase in rat brain has beenconfirmed and purified. Purified Sal synthase was identified by mass spectrometry and itsamino acid sequence was obtained. The synthase shows94.8%amino acid identity toubiquitin,so the gene of Salsolinol synthase could becloned by point mutation of ubiquitingene. In this paper, the gene of Salsolinol synthase was amplified, by PCR techniques, andcloned into prokaryotic pET28a vector and eukaryotic vector pcDNA4, then transferedinto the E.coli BL21and HEK cells respectively. Salsolinol synthase was expressedabundantly, the SDS-PAGE and Western-blot experients showed that the specific proteinmolecular weight is12KD, consistent with Salsolinol synthase protein molecular size.Purified Salsolinol synthase protein was obtained By affinity chromatography. Theenzymatic activity of Sal synthase was detected by liquid chromatography tandem massspectrometry (LC-MS/MS).The result showed that the overexpressed protein had bettercatalytic activity to condensate dopamine and acetaldehyde to Salsolinol than control group,indicating that Protein expressed by obtained sequence showed the enzymatic activity ofSal synthase.