Exploring PTDH–P450BM3 Variants for the Synthesis of Drug Metabolites

Abstract

The conversion of a series of pharmaceutical compounds was examined with three variants of cytochrome P450BM3 fused to phosphite dehydrogenase (PTDH) to enable cofactor recycling. Conditions for enzyme production were optimized, and the purified PTDH-P450BM3 variants were tested against 32 commercial drugs by using rapid UPLC-MS analysis. The sets of mutations (R47L/F87V/L188Q and R47L/F87V/L188Q/E267V/G415S) improved conversion for all compounds, and a variety of products were detected. Product analysis showed that reaction types included C-hydroxylation, N-oxidation, demethylation, and aromatization. Interestingly, enzymatic aromatization could occur independent of the addition of reducing coenzyme. These results identified new conversions catalyzed by P450BM3 variants and showed that a small set of mutations in the oxygenase domain could broaden both substrate range and reaction type.

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

“…Even though the fusion is rather large (155 kDa), expression is not compromised. The authors demonstrated the utility of PTDH–P450 by converting several pharmaceutical compounds to hydroxylated drug metabolites . A similar study was performed with a heterotrimeric P450 from Pseudonomas putida , in which it was fused to PTDH for self‐sufficient hydroxylations …”

“…The authors demonstrated the utility of PTDH-P450 by converting several pharmaceutical compounds to hydroxylated drug metabolites. [12,33] As imilar study was performed with ah eterotrimeric P450 from Pseudonomas putida, in whichi tw as fused to PTDH for self-sufficienth ydroxylations. [34] Enzyme fusion can be ag reat way to generate ac ofactor or cosubstrate forap articulare nzyme reaction.…”

“…By combining enzymes in this fashion, a single multifunctional biocatalyst can be produced that can catalyze a cascade of reactions. Remarkably, in a number of studies, evidence was found that the covalently tethered enzymes outperform the classic combination of the separate enzymes in some features such as enhanced expression, higher conversions of a cascade reaction, improved stability,, and improved catalytic activity . However, these promising observations are not consistent across studies, and generic methods to predictably design an effective bifunctional biocatalyst are still lacking.…”

“…Remarkably,i nanumber of studies, evidence was found that the covalently tethered enzymes outper-form the classic combination of the separatee nzymes in some features sucha se nhanced expression, [4,5] higherc onversions of ac ascade reaction, [4,[6][7][8][9] improved stability, [9a,c, 10] and improved catalytic activity. [9c, [10][11][12] However,t hese promising observations are not consistent across studies, andg eneric methods to predictably design an effective bifunctional biocatalyst are still lacking. Moreover, some studies find ad ecrease in activity as ac onsequence of enzyme fusion, thuss howingt hat fusing enzymes can be delicate.…”

Multi‐Functional Oxidase Activity of CYP102A1 (P450BM3) in the Oxidation of Quinolines and Tetrahydroquinolines

“…[11] We have adopted as imilar approach based on CYP102A1 variants found to have increased activity for unnatural substrate oxidation as aresult of the interaction between the heme iron and the axial water being weakened by anumber of mechanisms. [13] We report here the oxidation of THQs and quinolines by CYP102A1 variants with the aim to functionalize these building-block molecules selectively at as many positions as possible and thus provide synthetic handles to compounds with biological activity.F urther to C À Hb ond oxidation, we found that CYP102A1 displayed other oxidase activities, including desaturation, [14] aromatization, [15] and CÀCb ond formation reactions. [13] We report here the oxidation of THQs and quinolines by CYP102A1 variants with the aim to functionalize these building-block molecules selectively at as many positions as possible and thus provide synthetic handles to compounds with biological activity.F urther to C À Hb ond oxidation, we found that CYP102A1 displayed other oxidase activities, including desaturation, [14] aromatization, [15] and CÀCb ond formation reactions.…”