Discovery And Preliminary Catalytic Mechanism Of The Pictet-Spengler Enzyme

Pictet Spengler(P-S)reaction is always an old and new condensation reaction.It is still the most effective method to synthesizeβ-carboline and isoquinoline alkaloid and their related derivatives.Generally speaking,P-Sases refer to these enzymes that catalyze the P-S reaction which can catalyze the production of products with high stereochemical selectivity.The enzymes that catalyzed P-S reaction is rare in nature,so far only a few enzymes have been purified and identified.Based on this,a new P-S enzyme NscbB was discovered and identified by bioinformatics analysis and biochemical reaction in vitro.First of all,the amino acids sequence of McbB which has been identified as P-Sase was analyzed by BLAST analysis,and then located to NscbB through mining.Through phylogenetic analysis and multiple amino acid sequence alignment analysis,we preliminarily concluded that NscbB is also a P-Sase.Then,it was confirmed by biochemical experiments in vitro.Similar to McbB,NscbB can catalyze L-Trp and acetaldehyde in vitro to produce 1-acyl-3-carboxyl-β-carboline(1)and 1-acyl-β-carboline(2).This reaction does not need any cofactors,and the optimal reaction temperature range is from 28℃-37℃,and the optimal pH value is 7.0.Thus,it was confirmed that NscbB is a new P-Sase.It was found that NscbB/Glu97 completely lost the enzymatic activity in the point mutation experiment,which indicated that Glu97 was a key amino acid residue in the catalytic reaction of NscbB.The biochemical reaction revealed that NscbB can catalyze L-tryptophan with a K_m of 89.64±8.69μM and methylglyoxal with a K_m of 147.70±16.38μM.Finally,the resulting nscbB gene heterologous expression in E.coli BL21(DE3)to bioproduce twobC skeletons,1-acetyl-3-carboxy-β-carboline(1,5.5 mg/L)and1-acetyl-β-carboline(2,3.1 mg/L)in vivo,in 16 hours of fermentation confirmed the heterologous biosynthesis potential and production capacity of NscbB.In conclusion,a novel P-S enzyme,NscbB,was discovered and identified by bioinformatics analysis combined with biochemical reaction in vitro and heterologous expression in vivo.Its enzymatic properties were characterized preliminarily and the key amino acid residue was identified.NscbB is new functional and practical microbial PSase that could be utilized for future bioactivebC alkaloids exploitation and development.The research work providing an appropriate gene and enzyme tool for homologous mining from nature and constructing new chemical entities library by gene mining,combinatorial biosynthesis and synthetic biology technology,and also provides the foundation for the discovery and systematic research of P-Sases in the future.