4' Deoxytpe Chalcone Synthase from Scutellaria baicalensis

黄芩是我国传统中药中的一种重要成分。最近，其抗病毒活性的主要成分被鉴定为黄芩素，抗流感病毒活性的主要成分被鉴定为5，7，4 ' -羟基-8-甲氧基黄酮。从生物合成的角度来看，4' -氧和4' -脱氧型黄酮类化合物在同一植物中的共存也是非常有趣的。由于在查尔酮形成后在4'位脱氧似乎是不可能的，因此可以假设在查尔酮合酶（CHS）反应中两种类型的查尔酮的加工。为了阐明一个CHS是否同时利用4-香豆酰-CoA和肉桂酰-CoA作为底物，或者存在两个不同的CHS，我们尝试从该植物中克隆CHS的cDNA。制备逆转录的cDNA ...更多信息 d从S. baicalensis并用作模板。从S. RT-PCR检测黄芩（P1-P5）。构建含有这些基因的6-组氨酸残基的表达质粒，以研究cDNA是否编码肉桂酰CoA特异性CHS。E.将P1和P4分别转化大肠杆菌，经高效液相色谱分析，得到了松属素和柚皮素，并对异源表达的两种酶（P1和P4）进行了纯化和动力学研究。通过两次Ni-螯合亲和层析和随后的DEAE离子交换层析纯化所得蛋白溶液。经3步纯化后，酶比活力分别提高了50倍和175倍，分别为369 pkat/mg和26 pkat/mg，纯化后的P1酶对p-香豆酰辅酶A的最适pH为8.0，对肉桂酰辅酶A的最适pH为7.0 ~ 7.5。对于p-香豆酰-CoA，观察到的P1的Km值为36.1μM，对于肉桂酰-CoA，观察到的P1的Km值为54.7μM。而P4的最适pH8.0和pH7.5分别为对香豆酰辅酶A和肉桂酰辅酶A。对香豆酰辅酶A和肉桂酰辅酶A的Km值分别为3.5 μM和2.6 μM，表明P4基因编码的酶对肉桂酰辅酶A具有选择性，可能参与了脱氧型黄酮的合成。与P1的kcat值相比，kcat值较低，这可能是由于在Ala 58（通常为Gln）处引入了异常的氨基酸残基，该氨基酸残基可能是由错误的序列分析引入的。P4是，虽然序列是可疑的，仍然是一个很好的例子，进行有趣的底物特异性。少

Scutellaria baicalensis (Labiatae) has been used in Chinese traditional medicine for a long time. Recently, its principle for anti-viral activity has been identified as baicalein, and for anti-influenza virus activity3 as 5,7,4' -hydroxy-8-methoxy-flavone. Coexistence of 4' -oxy and 4' -deoxy type flavonoids in the same plant is very interesting from biosynthetic point of view as well. Since deoxygenation at 4' position after chalcone formation seems very unlikely, elaboration of two types of chalcones in chalcone synthase (CHS) reaction could be assumed. In order to clarify whether one CHS can utilize both 4-coumaroyl- and cinnamoyl-CoA as substrates, or two distinct CHSs specific for 4-coumaroyl- and cinnamoyl-CoA as priming substrate are occurring, cDNA cloning of CHS from this plant has been attempted.An aerial part of this plant contains flavonoids lacking 4' -hydroxyl group, the young leaves of the plant were chosen for extraction of the mRNA. Reverse transcribed cDNA was prepare … More d from the total RNA of the leaves of S. baicalensis and used as a template. Five CHS genes were cloned from S. baicalensis by RT-PCR (P1-P5). The expression plasmids bearing 6-histidine residues of those genes were constructed to investigate whether the cDNA codes CHS specific for cinnamoyl-CoA. The extracts of E. coli transformed with expression vectors harboring P1 and P4 respectively produced pinocembrin and naringenin that were identified by HPLC analysis.Then heterologously expressed both enzymes (P1 and P4) were purified to serve for kinetic experiments. The resulted protein solutions were purified through twice Ni-chelete affinity chromatography and following DEAE ion exchange chromatography. The 3 step purification afforded 50 and 175-fold increase in specific activity of those enzyme fractions at 369 pkat/mg and 26 pkat/mg respectively.The purified enzyme of P1 showed optimum pH8.0 with ρ-coumaroyl-CoA and between pH7.0 and 7.5 with cinnamoyl-CoA for naringenin and pioncembrin production. The observed Km values of P1 were 36.1μM for ρ-coumaroyl-CoA and 54.7μM for cinnamoyl-CoA. In contrast, the enzyme of P4 showed optimum pH8.0 with ρ-coumaroyl-CoA and pH7.5 with cinnamoyl CoA for naringenin and pinocembrin production. The observed Km values of P4 were 3.5 μM for ρ-coumaroyl-CoA and 2.6 μM for cinnamoyl-CoA.This result suggests that the enzyme encoded P4 showing preference to cinnamoyl CoA possibly is involved in biosynthesis of deoxy type flavonoid, however. The inferior kcat value comparing to that of P1's may be caused by the extraordinary amino acid residue at Ala58 (normally Gln) that plausibly was introduced by wrong sequence analysis. The P4 is, though the sequence is dubious, remains a good example that carries interesting substrate specificity. Less