4' Deoxytpe Chalcone Synthase from Scutellaria baicalensis

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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