Design of artificial enzymes

The residues at the substrate binding site of the cytochrome P450 located above the heme center plays the key role in anchoring the substrate in a specific orientation facilitating reaction of the substrate with oxygen catalyzed by the mono-oxygenase enzyme:

(R_1 R_2 R_3)C-H+O_2+2e^-+2H^(+ )⋯⋯→(R_1 R_2 R_3)C-OH +H_2 O

The group is carrying out detailed molecular docking studies to identify the residues that may be involved in anchoring a particular type of substrate. Site-specific mutations at those residues are used to create recombinant enzyme that can bind and catalyse reaction of the substrate of choise.

The CuA centre in the subunit II of the respiratory enzyme cytochrome c oxidase binds the metal ions through a loop connecting two anti-parallel beta-sheets in the protein and only one ligand to the metal ion is provided by a histidine from another loop of the protein. We are designing a series of peptides containing Histidine, Methionine, and Cysteine as ligands to the copper ion and studying their copper binding properties using different spectroscopic and kinetic methods. A small pentapeptide motif has been identified that was indicated to be the smallest unit of the peptide loop that might bind the copper ion. The binding of copper ion causes change in the backbone conformation of the peptide loop which might trigger movement of the metal ion binding loop towards the center and thus facilitating transfer of copper ion to the protein. This might provide a molecular mechanism of conformationally coupled sequential incorporation of two copper ions forming the dithiolato-bridged purple copper complex in the protein.

Ref: (Manuscript under publication) Dwaipayan Dutta Gupta, Imon Mandal, Chandrani Nayak, Shambhu Nath Jha, Ravindra Venkatramani, Dibyendu Bhattacharyya and Shyamalava Mazumdar

Design of novel copper binding peptides based on the metal ion binding loop of the CuA centre of cytochrome c oxidase

Three stable copper complexes of peptides derived from the copper ion binding loop of the subunit II of cytochrome c oxidase have been prepared and characterized by various spectroscopic techniques. These stable copper complexes of peptides were found to exhibit cysteine, histidine and/or methionine ligation, which has predominant σ-contribution in the Cys–Cu charge transfer. The copper(II) peptide complexes showed type-2 EPR spectra, which is uncommon in copper–cysteinate complexes. UV-visible spectra, Raman and EPR results support a tetragonal structure of the coordination geometry around the copper ion. The copper complex of the 9-amino acid peptide suggested the formation of a ‘red’ copper center while the copper complexes of the 12- and 11-amino acid peptides showed the formation of a ‘green’ copper center. The results provide insights on the first stable models of the copper complexes formed in the peptide scaffold that mimic the mono-nuclear copper bound protein intermediates proposed during the formation of the binuclear Cu2S2 core of the enzyme. These three copper complexes of peptides derived from the metal ion binding loop of the CuA center of the subunit II of cytochrome c oxidase showed novel spectroscopic properties which have not so far been reported in any stable small complex.

Ref: Mono-nuclear copper complexes mimicking the intermediates for the binuclear copper center of the subunit II of cytochrome oxidase: a peptide based approach, Dwaipayan Dutta Gupta, Dandamudi Usharani and Shyamalava Mazumdar, Dalton Trans., 2016, 45, 17624–17632

Superposed structures of the metal ion binding loop of HoloCuA (green & cyan, PDB code: 2CUA) and apo-CuA (magenta, PDB code: 2LLN) showing the copper ion binding site of the protein.

Enzymatic mono-oxygenation of unsaturated fatty acids by thermostable cytochrome P450 enzyme

CYP175A1 is a thermophilic P450 with high potential to invoke as an industrially viable biocatalyst. However, very little is known about the natural substrate that can undergo biotransformation in the enzyme pocket. The crystal structure of CYP175A1 was found to be closely related to its mesophilic analogue P450 BM-3, which is a fatty acid metabolizing enzyme. Our studies had revealed that CYP175A1 catalyzes regioselective mono-oxygenation of different monounsaturated fatty acids depending upon the position and stereochemistry of the double bond in the substrate. We showed that polyunsaturated fatty acids (arachidonic acid, linoleic acid, α-linolenic acid & 𝛾-linolenic acid) can also be oxygenated by the enzymatic action of CYP175A1 although the enzyme did not show any detectable activity on the corresponding saturated analogues (arachidic acid and stearic acid). The product analyses show that unlike monounsaturated fatty acids, polyunsaturated fatty acids undergo mono- as well as di-oxygenation reactions. Further, with the increase in unsaturation of the fatty acid the yield of mono-oxygenated product improved. The product analyses show that the regioselectivity of these oxygenation reaction is tightly regulated by the number and position of the double bonds in the fatty acids. Molecular docking calculations suggested that “U”-type conformations of the polyunsaturated fatty acids are particularly responsible for their binding at the enzyme pocket, and that is also consistent with the observed regioselectivity in the oxygenation reaction

Ref: Regioselective Oxygenation of Polyunsaturated Fatty Acids by the Thermostable P450 from Thermus thermophilus HB27,
Shibdas Banerjee, Dwaipayan Datta Gupta, and Shyamalava Mazumdar, Current Biotechnology, (2015), 4(3): 345 – 356

Superposition of CYP175A1 (red; PDB entry 1N97) and P450 BM3 (green; PDB entry 1FAG) using SuperPose Version 1.0. (b) Docking model of the linoleic acid (LA) in the active site of CYP175A1 obtained by the GOLD program. Three substrate (LA) conformers in the enzyme pocket (above the heme) with the top three binding scores have been presented here as the possible modes of binding of the substrate to the enzyme pocket. In all these there conformations the ethylenic double bonds (shown in green colors) are populated nearer to heme iron and thus they are the probable sites susceptible to oxygenation.

Role of substituents on the reactivity and product selectivity in reactions of naphthalene derivatives catalyzed by the orphan thermostable cytochrome P450, CYP175A1

The thermostable nature of CYP175A1 enzyme is of potential interest for the biocatalysis at ambient temperature or at elevated temperature under environmentally benign conditions. Although little is known about the substrate selectivity of this enzyme, the biocatalytic activities of CYP175A1 on different substituted naphthalenes have been studied in oxidative pathway, and the effect of the substituent on the reaction has been determined. The enzyme first acts as a peroxygenase to convert these substituted naphthalenes to the corresponding naphthols, which subsequently undergo in-situ oxidative dimerization to form dyes of different colors possibly by the peroxidase-type activity of CYP175A1.

The product analyses and kinetic measurements suggested that the presence of electron releasing substituent (ERS) in the substrate enhanced the substrate conversion, whereas the presence of electron withdrawing substituent (EWS) in the substrate drastically reduced the substrate conversion. The position of the ERS in the substrate was also found to play an important role in the transformation of the substrate. The results further demonstrate that mutation of the Leu80 residue to Phe enhances the reactivity of the enzyme by favoring the substrate association in the active site. The observed rates of the enzymatic oxygenation reaction of the substituted naphthalenes followed the Hammett correlation of substituent effect, supporting aromatic electrophilic substitution mechanism catalyzed by the cytochrome P450 enzyme.

Ref: Role of substituents on the reactivity and product selectivity in reactions of naphthalene derivatives catalyzed by the orphan thermostable cytochrome P450, CYP175A1 Shibdas Banerjee, Sandeep Goyal and Shyamalava Mazumdar, Bioorganic Chemistry (2015), 62, 94-105