Biotransformation of Flavone by CYP105P2 from Streptomyces peucetius

Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, SunMoon University, Asansi 336-708, Ko reaReceived: January 26, 2012 / Revised: April 9, 2012 / Accepted: April 10, 2012Biocatalytic transfer of oxygen in isolated cytochromeP450 or whole microbial cells is an elegant and efficientway to achieve selective hydroxylation. Cytochrome P450CYP105P2 was isolated from Streptomyces peucetiusthat showed a high degree of amino acid identity withhydroxylases. Previously performed homology modeling,and subsequent docking of the model with flavone,displayed a reasonable docked structure. Therefore, inthis study, in a pursuit to hydroxylate the flavone ring,CYP105P2 was co-expressed in a two-vector system withputidaredoxin reductase (camA) and putidaredoxin (camB)from Pseudomonas putida for efficient electron transport.HPLC analysis of the isolated product, together with LC-MS analysis, showed a monohydroxylated flavone, whichwas further established by subsequent ESI/MS-MS. Asuccessful 10.35% yield was achieved with the whole-cellbioconversion reaction in Escherichia coli. We verifiedthat CYP105P2 is a potential bacterial hydroxylase.Keywords: Cytochrome P450, flavone, redox partner, Streptomycespeucetius, whole-cell biotransformationThe reactions catalyzed by microorganisms involvingflavones include oxidation, reduction, conjugation, anddeglycosylation [5]. Numerous flavonoids biotransformedby microbes to improve their pharmaceutical activitieshave been studied. Recently, Escherichia coli has beenidentified as a suitable host for whole-cell bioconversion,although a few examples of the use of Streptomycetesstrains for biotransformation have also been reported [33].Cytochrome P450s (CYP450s) are highly attractive biocatalysts,as they are responsible for selective hydroxylation ofaromatic compounds, which is among the most challengingchemical reactions. These enzymes have gained steadilyincreasing attention during recent years, particularlybecause of the use of hydroxylated aromatics as precursorsfor pharmaceuticals. However, the low activity and themulticomponent nature of these enzymes often result inpoor productivities. Therefore, co-expression is a powerfultool for the optimization of whole-cell CYP450 biocatalysis.The co-expression of genes encoding the redox partners ofthe CYP450 is necessary when the host cell lacks suchelectron transfer proteins or when the activity with anendogenous system is low [25]. The flavoprotein in thissystem, putidaredoxin reductase (CamA), contains FADand is a strictly NADH-dependent ferredoxin reductaselacking the NADP-binding sequence [8, 23]. The ironsulfur protein, putidaredoxin (CamB), belongs to the [2Fe–2S] ferredoxin group and plays the role of an electronshuttle transferring the two electrons one at a time fromputidaredoxin reductase to P450cam [7, 8].Three classes of substrates, namely 7-ethoxycoumarin,fatty acids, and flavonoids [34], were selected to study thehydroxylating activity of CYP450s from Streptomycespeucetius by exploring their substrate specificity. Out of 23CYP genes present in the genome of S. peucetius, fourCYPs (CYP105F2, CYP105P2, CYP107N3, and CYP157C4)that were expressed as soluble proteins exhibited remarkableCO-difference spectra and were chosen as candidates forbioconversion of the substrates. CYP105F2 was previouslyverified for hydroxylation of oleondomycin [30]. Our initialattempt to identify possible substrates for CYP105P2 bybinding studies and homology modeling turned out to besignificant for the flavonoids (flavone, flavanone, quercitin,and naringenin) class of substrates [16]. Among othersubstrates such as macrolides, 7-ethoxycoumarin used fordocking, flavone was best en suite in the constructedmodel with ring-B directed towards the reaction center;iron [16]. Intrinsically, P450 enzymes are not very active;they exhibit poor stability in their isolated form. Therefore,owing to this fact, we co-expressed CYP105P2 with theredox partner camA/camB in E. coli that readily generatedthe monohydroxylated flavone.Furthermore, the structure-based pharmaceutical activationof flavones has led to extensive research of biotransformation