查尔酮合酶蛋白表达技术、结构、活性和进化

查尔酮合酶(CHS)是类黄酮途径的首步关键酶,参与合成所有类黄酮和异黄酮物质,参与决定多种植物重要性状。NCBI已有2917条CHS序列登录和释放。单个物种基因组中的CHS普遍由基因家族编码,通过基因加倍而产生。CHS蛋白的表达技术目前均是基于大肠杆菌的原核表达,多采用Novagen公司的pET载体系统,最通行参数为37℃条件下1 mmol/L IPTG诱导3h,表达蛋白普遍采用His-Tag进行亲和纯化,采用质谱或免疫分析技术进行身份检测,通过对生化反应底物和产物的HPLC检测可以精确分析酶活。苜蓿等植物的CHS蛋白已完成X射线晶体衍射研究,获得了三维结构和活性位点构象的初步解析,并与植物Ⅲ型PKS超家族中的其它酶类进行了比较。CHS蛋白的催化活性中心含有一个Cys-His-Asn三联体,另外CoA结合通道和内部的起始/延伸/环化腔关系到CHS蛋白的底物特异性及聚酮化合物链延伸的长度。植物Ⅲ型PKS超家族中,其它酶的结构骨架和催化方式与CHS相似,但活性位点残基的变化可能会造成活性中心结构的改变,进而产生底物特异性、催化能力和产物种类的显著变化,是蛋白水平进化的根本动力。

Protein Expression Technologies, Structure, Activity and Evolution of Chalcone Synthase

The chalcone synthase(CHS) was the first-step key-enzyme of flavonoid pathway,participating in biosynthesis of all flavonoid and isoflavonoid compounds and determining many important plant traits.In NCBI,2917 CHS sequences were submitted and released.Within the genome of a single species,general CHS was encoded by a gene family which was generated by gene duplication.Currently CHS protein was mainly expressed in prokaryotic Escherichia coli using pET vector system from Novagen Company.The popular optimum parameters were to inoculate 3 h by 1mmol/L IPTG under 37 ℃.The expressed CHS protein was generally purified through His-Tag affinity purification,identified by mass chromatography or immunoassay,and activity-tested by HPLC analysis of the substrates and the products.X-ray crystal diffraction analysis was performed on CHS proteins from Medicago sativa etc.,and the three-dimensional structures of the protein and the active site were dissected,which were compared with those of other enzymes from plant type Ⅲ PKS superfamily.The catalytic center of CHS contained a Cys-His-Asn triad,and the CoA-binding tunnel and inner initiation/elongation/cyclization cavity were crucial for substrate specificity and elongation length of polyketides of CHS.Within plant type Ⅲ PKS superfamily,other enzymes shared similar backbone and catalytic manner with CHS,but the change of active-site residues might bring to the change of the structure of active center,thus cause significant change in substrate specificity,catalytic activity and product type,which was the basic force for protein-level evolution.