Design of artificial enzymes

The Residues at the substrate binding site of the cytochrome P450 located above the hemi centre plays the keys role in anchoring the substrate in a specific orientation facilitating reaction of the substrate with oxygen catalysed by the mono-oxygenase enzymes:

(R1 R2 R3) C – H + O2 + 2e- + 2H+ …… …-> (R1R2R3)C – OH + H2O

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

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.

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

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.

Design of novel copper binding peptides based on the metal ion binding loop of the CuA centre of cytochrome c oxidase
The conformational properties and stability of the metal active center are important for the biochemical function of metal enzymes and proteins. We have determined the specific contributions of hydrogen-bonding, ionic and hydrophobic interactions on the stabilization of the active site in the plant peroxidase and showed that the high stability of the enzyme against hydrogen peroxidase arises due to extensive hydrogen bonding and salt-bridges surrounding the metal center in the enzyme. Removal of the metal ion prosthetic group was shown to have a drastic effect on the structure of the enzyme and Our studied have also shown existence of multiple conformational sub-states in the apo-protein of the peroxidase in solution.

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

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.

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

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

Reversible inactivation of cytochrome P450 by alkaline earth metal ions: Auxiliary metal ion induced conformation change and formation of inactive P420 species in CYP101 Soumen K. Manna, Shyamalava Mazumdar
J. Inorg. Biochem. (2008)5-6, 1312 – 1321

Effect of Polar Solvents on the Optical Properties of Water-dispersible Thiol-Capped Cobalt Nanoparticles
Nusrat J M Sanghamitra and Shyamalava Mazumdar
Langmuir (2008) 24 (7), 3439 – 3445

Conformational dynamics coupled to protonation equilibrium at the CuA site of T. thermophilus: Insights into the origin of thermostability
Nusrat J M Sanghamitra and Shyamalava Mazumdar
Biochemistry (2008) 47, 1309 – 1318

Thermostability of proteins: Role metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus
A. Sujak, Nusrat J. M. Sanghamitra, O. Maneg, B. Ludwig and Shyamalava Mazumdar
Biophys. J. (2007) 93(8), 2845–2851

Effect of Alcohols on Binding of Camphor to Cytochrome P450cam: Spectroscopic and Stopped Flow Transient Kinetic Studies
R. Murugan and Shyamalava Mazumdar
Arch. Biochem. Biophys. (2006) 455(2), 154-162

Role of Threonine 101 on the stability of the heme active site of cytochrome P450cam: Multi-wavelength Circular Dichroism Studies
Soumen Kanti Manna and Shyamalava Mazumdar
Biochemistry (2006) 45(42), 12715 – 12722

Structure of the heme centre and its redox properties in the C357M mutant of cytochrome P450cam
R. Murugan and S. Mazumdar
ChemBioChem, (2005) 6, 1204-1211

Direct Correlation of the Crystal Structure of Proteins with the Maximum Positive and Negative Charge-States of Gaseous Protein Ions Produced by Electrospray Ionization
Halan Prakash and S. Mazumdar
J. Am. Soc. Mass Spectrom. (2005) 16, 1409-1421

Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants
R. Murugan and S. Mazumdar
J. Biol. Inorg. Chem. (2004) 9, 477-488

Stabilization of Partially Folded States of Cytochrome C in Aqueous Surfactant:
Effects of Ionic and Hydrophobic Interactions
K. Chattopadhyay and S. Mazumdar
Biochemistry (2003) 42, 14606 – 14613

Redox Linked Conformational Change in Bovine Heart Cytochrome c Oxidase: Picosecond Time-resolved Fluorescence Studies of the Cyanide Complex
T. K. Das and S. Mazumdar
Biopolymers: Biospectroscopy (2000) 57, 316-322

Structural and Conformational Stability of Horseradish Peroxidase: Effect of Temperature and pH
K. Chattopadhyay and S. Mazumdar
Biochemistry, (2000) 39(1), 263-270.

Direct electrochemical oxidation of horseradish peroxidase: Cyclic Voltammetric and spectroelectrochemical studies
K. Chattopadhyay and S. Mazumdar
New J. Chem., (1999) 2, 137-139.