Bioinformatics Analysis,Heterologous Fusion Expression And In Vitro Catalytic Studies Of CYP75A110 Phenol-coupled Enzyme

Plant isoquinoline alkaloids(PIAs)are a class of natural products with diverse structures and medicinal activities.Phenol coupling reaction is not only an indispensable step in the synthesis of PIAs framework,but also one of the important problems in organic synthesis reactions,because both stereoselectivity and regioselectivity need to be taken into account.Since phenol-coupling enzymes can provide an anisotropic environment for the coupling reaction to achieve regioselectivity and stereoselectivity,they are considered to be the preferred catalysts for selective intermolecular/intraphenol coupling.In this paper,the CPY75A110phenol-coupled enzyme derived from Gloriosa superba was used as the research object,aiming to express the soluble phenol-coupled enzyme in a heterologous system,and to explore the enzymatic synthesis of the PIA acetylcholine inhibitor galantamine.The subsequent molecular modification and heterologous expression of plant P450phenol-conjugating enzymes and their application in biosynthesis lay the foundation.In this paper,using bioinformatics methods and techniques,CPY75A110phenol-conjugated enzyme from Gloriosa superba was analyzed for its amino acid physicochemical properties,hydrophilicity/hydrophobicity,transmembrane region and signal peptide.The results showed that CPY75A110 was unstable,Hydrophobic protein containing a signal peptide and a transmembrane domain.The secondary structure of CYP75A110 was predicted,and the results showed that its secondary structure contained 7 basic types,namely β-sheet,β-hairpin,β-protrusion,β-sheet,α-helix,β-turn,and γ-turn.β-sheets are formed by parallel arrangement of β-sheets.The β-turn,γ-turn and β-hairpin are close to each other to form a super-secondary structure,which forms the nucleation center during protein folding.The homology modeling of CPY75A110 was carried out,and the pull-type conformation map analysis showed that the model was reasonable and reliable.The CPY75A110 binding pocket was predicted to contain a large heme center(HEM)and a cysteine ??at position 432.Molecular docking of(S)-Autumnaline and navidine precursors with CYP75A110 showed that both ligands can enter the receptor’s binding pocket and undergo hydrogen bonding,π-π stacking and hydrophobicity with the receptor’s amino acid residues use each other.Among them,Pro357,Leu362,and Phe425 are the amino acid residues that CYP75A110 interacts with these two substrates.It is speculated that these three amino acids are the key amino acids that affect the catalytic activity of the phenol-coupled enzyme CPY75A110.CYP75A110 was genetically modified and optimized.After truncation of the N-terminal signal peptide and codon optimization,it was fused in series with the cytochrome reductase Rhf RED to construct the p MAL-c5x-CYP75A110-Rhf RED expression vector,which was expressed in E.coli Rosetta(DE3)achieved soluble expression of the CYP75A110-Rhf RED fusion enzyme.The genome was extracted from Bacillus subtilis(Bacillus subtilis 168),and the glucose dehydrogenase(GDH)gene fragment was obtained by primer design and PCR amplification,and the p ET28a-GDH expression vector was constructed.Soluble GDH.Using the glucose dehydrogenase-glucose(GDH-Glucose)coenzyme regeneration system,a dual-enzyme catalytic system of GDH and CYP75A110-Rhf RED in vitro was constructed to successfully catalyze the substrate 2N-(5-hydroxy-4-methoxybenzyl)-4-Hydroxyphenethylmethylamine obtains navidine,the key intermediate of galantamine.After codon optimization of carbonyl reductase SAR in morphine biosynthesis,p ET28a-SAR expression vector was constructed,and soluble SAR was obtained in E.coli BL21(DE3).Using the glucose dehydrogenase-glucose(GDH-Glucose)coenzyme regeneration system,an in vitro dual-enzyme catalytic system for GDH and SAR was constructed,and it was verified by mass spectrometry that it could catalyze navidine to obtain galantamine.