Vol.:(0123456789) 1 3 The Protein Journal https://doi.org/10.1007/s10930-022-10084-3 Enhanced Substrate Specifcity of Thermostable Cytochrome P450 CYP175A1 by Site Saturation Mutation on Tyrosine 68 Srabani Karmakar 1,2  · Sudip Kumar Nag 2  · Mohd Taher 1  · Bharat T. Kansara 1  · Shyamalava Mazumdar 1 Accepted: 13 October 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022 Abstract Thermostable cytochrome P450 (CYP175A1) cloned from Thermus thermophilus shows mid-point unfolding tempera- ture (Tm) of 88 °C (361 K) along with high thermodynamic stability making it a potential industrially viable biocatalyst. Molecular docking analyses, and structural superposition with steroidogenic and fatty acid metabolizing cytochrome P450 s suggested that the tyrosine 68 may have important role in binding as well as metabolism of substrates by the enzyme. Site-saturation mutation of the tyrosine 68 residue was carried out and several unique mutations were obtained that were properly folded and showed high thermostability. We investigated the efects of variation of the single residue, Tyr68 at the substrate binding pocket of the enzyme on the substrate specifcity of CYP175A1. Screening of the mutant colonies of CYP175A1 obtained after saturation mutagenesis of Tyr68 using saturated fatty acid, myristic acid and poly unsaturated fatty acids showed that the Y68K had notable binding and catalytic activity for mono-oxygenation of the saturated fatty acid (myristic acid), which had no major detectable binding afnity towards the WT enzyme. The Y68R mutant of CYP175A1, on the other hand was found to selectively bind and catalyse reaction of cholesterol. The wild type as well as both the mutants of the enzyme however bind poly unsaturated fatty acids. The results thus show that saturation mutation of a single amino acid at the substrate binding pocket of the thermostable cytochrome P450 could induce sufcient changes in the substrate binding pocket of the enzyme that can efciently change substrate specifcity of the enzyme. Keywords Cytochrome P450 · CYP175A1 · Substrate binding · Mass spectrometry · Circular dichroism 1 Introduction The cytochromes P450’s forms a superfamily of heme monooxygenases that catalyse heterolytic cleavage of molecular oxygen for insertion of a single oxygen atom at a specifc site of the reducing substrate [1, 2]. The substrate binds at the core of the enzyme in the distal side of the heme center anchored to a cysteine residue [2]. A wide variety of reactions, including aromatic and aliphatic hydroxylation, N oxidation and epoxidation of double bonds are catalysed by these enzymes. Thus, these enzymes have great potential for biotechnological application as catalysts for synthesis of industrially important molecules, and for degradation of toxic substances e.g., polyaromatic hydrocarbons. Extensive studies have earlier demonstrated that site-specifc modifca- tion of the active site can be used to alter the substrate speci- fcity of the enzyme [2–4]. However, most of these studies were based on cytochrome P450 isoenzymes from meso- philic sources such as cytochrome P450BM3, cytochrome P450cam etc. Studies of thermostable cytochrome P450 enzymes can thus help in developing suitable enzymes with viable applications [3, 4]. Earlier studies have shown that mutation of the thermostable enzyme CYP119 can catalyse fatty acid hydroxylation [5], and CYP175A1 from Thermus thermophilus HB27 can catalyse hydroxylation of beta-car- otene [3, 6–10]. The natural redox partners for CYP175A1 was identifed and isolated recently [11]. Protein engineer- ing studies have developed various site-specifc mutants of CYP175A1 with diferent functional properties [2, 12–15]. The double mutant Q67G/Y68I of CYP175A1 was shown to hydroxylate testosterone [11]. CYP175A1 was also shown to * Srabani Karmakar snat14@gmail.com * Shyamalava Mazumdar shyamal@tifr.res.in 1 Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India 2 Department of Biotechnology, Techno India University West Bengal, EM-4 Sector V, Salt Lake, Kolkata 700091, India