Computationally guided bioengineering of the active site, substrate access pathway, and water channels of thermostable cytochrome P450, CYP175A1, for catalyzing the alkane hydroxylation reaction.

Understanding structure-function relationships in proteins is pivotal in their development as industrial biocatalysts. In this regard, rational engineering of protein active site access pathways and various tunnels and channels plays a central role in designing competent enzymes with high stability and enhanced efficiency. Here, we report the rational evolution of a thermostable cytochrome P450, CYP175A1, to catalyze the C-H activation reaction of longer-chain alkanes.

Mechanistic evaluation of chromium bioremediation in Acinetobacter junii strain b2w: A proteomic approach.

Growing industrialization and unchecked release of industrial waste, including heavy metals have resulted in disastrous effects on environment. Considering the problem of heavy metal pollution, the present research was designed to study the bioremediation of chromium, a highly toxic and prominent heavy metal pollutant by Acinetobacter junii strain b2w isolated from the Mithi river, Mumbai, India. The bacterial isolate could grow without affecting its growth kinetics up to a concentration of 200 ppm of chromium and showed resistance towards 400 ppm of chromium.

Antibacterial Effects of ZnO Nanodisks: Shape Effect of the Nanostructure on the Lethality in Escherichia coli.

The role of the shape of the nanostructure on the antibacterial effects of ZnO nanodisks has been investigated by detailed mass spectrometry-based proteomics along with other spectroscopic and microscopic studies on E. coli. The primary interaction study of the E.

Controlled Uptake of an Iridium Complex inside Engineered apo-Ferritin Nanocages: Study of Structure and Catalysis.

The effect of the mutation at the core of the ferritin nanocage (apo-rHLFr) on the uptake of IrCp* has been investigated by structural and spectroscopic methods. Site-specific mutations of two polar residues viz., Asp38 and Arg52 were investigated.

Covalent conjugation of single-walled carbon nanotube with CYP101 mutant for direct electrocatalysis.

Covalent linkage between the single-walled carbon nanotube (SWCNT) and CYP101 through a specific site of the enzyme can provide a novel method of designing efficient enzyme electrodes using this prototype cytochrome P450 enzyme. We have chemically modified the SWCNT with linker 4-carboxy phenyl maleimide (CPMI) containing maleimide functional groups. The enzyme was covalently attached on to the SWCNT through the maleimide group of the linker (CPMI) to the thiolate group of the surface exposed Cys 58 or Cys 136 of the CYP101 forming a covalently immobilized protein on the nanotube.

Unveiling the urease like intrinsic catalytic activities of two dinuclear nickel complexes towards the in situ syntheses of aminocyanopyridines.

Designing metal complexes as functional models for metalloenzymes remains one of the main targets in synthetic bioinorganic chemistry. Furthermore, the utilization of the product(s) derived from the catalytic reaction for subsequent organic transformation that occurs in biological systems is an even more difficult challenge for biochemists. Urease, the most efficient enzyme known, catalyzes the hydrolysis of urea and it contains an essential dinuclear Ni cluster in the active site.

Kinesin associated protein, DmKAP, binding harnesses the C-terminal ends of the Drosophila kinesin-2 stalk heterodimer.

The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known.

The structural dynamics of the kinesin-2 stalk heterodimer and its biological relevance.

Association between two motor subunits through the rod/stalk domain enables molecular motors to walk processively on protein filaments. Previous studies suggested that structural flexibility in the coiled-coil stalk of kinesins is essential for processive runs. The stalk of heterotrimeric kinesin-2, a comparatively less processive motor, is unstable at ambient temperature.

The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis.

The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear.

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.

A molecular Fe-complex as a catalyst probe for in-gel visual detection of proteins via signal amplification.

We report the use of a molecular peroxidase mimic biuret-Fe-TAML for chemoselective labeling of proteins and the subsequent visual detection (<0.1 pmoles) of the conjugate in a polyacrylamide gel by catalytic signal amplification. Use of this probe in activity based protein profiling (ABPP) of serine proteases is also demonstrated.

Mechanism of copper incorporation in subunit II of cytochrome C oxidase from Thermus thermophilus: identification of intermediate species.

Detailed spectroscopic and kinetic studies of incorporation of copper ion in the wild type (WT) and the D111AA (AA = K, N, or E) mutants of the metal ion binding site of the soluble fragment of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) showed the formation of at least two distinct intermediates. The global analyses of the multiwavelength kinetic results suggested a four-step reaction scheme involving two distinct intermediates in the pathway of incorporation of copper ions into the apoprotein forming the purple dinuclear CuA. An early intermediate similar to the red copper binding proteins was detected in the WT as well as in all the mutants.

Thermodynamic effects of the alteration of the axial ligand on the unfolding of thermostable cytochrome C.

The role the axial methionine plays in the conformational properties and thermostability of the heme active site has been investigated with the help of site-specific mutations at the axial Met69 position with His (M69H) and Ala (M69A) in thermostable cytochrome c(552) from Thermus thermophilus. Detailed circular dichroism, direct electrochemistry, and other spectroscopic studies have been employed to investigate the thermally induced and GdnHCl-induced unfolding properties of the heme active site of the wild type and the mutants of cytochrome c(552). We observed an unusually high thermodynamic and thermal stability of the M69A mutant compared to that of wild-type cytochrome c(552).

Biochemical and molecular dynamic simulation analysis of a weak coiled coil association between kinesin-II stalks.

Definition: Kinesin-2 refers to the family of motor proteins represented by conserved, heterotrimeric kinesin-II and homodimeric Osm3/Kif17 class of motors.

Background: Kinesin-II, a microtubule-based anterograde motor, is composed of three different conserved subunits, named KLP64D, KLP68D and DmKAP in Drosophila. Although previous reports indicated that coiled coil interaction between the middle segments of two dissimilar motor subunits established the heterodimer, the molecular basis of the association is still unknown.

Oxygenation of monoenoic fatty acids by CYP175A1, an orphan cytochrome P450 from Thermus thermophilus HB27.

The catalytic activity of CYP175A1 toward monooxygenation of saturated and monounsaturated fatty acids of various chain lengths (C16-C24) has been investigated to assess the enzymatic properties of this orphan thermostable cytochrome P450 enzyme. The results showed that the enzyme could catalyze the reaction of monounsaturated fatty acids but not of saturated fatty acids. The product analyses using ESI-MS and GC-MS revealed an important regioselectivity in the CYP175A1 catalyzed monooxygenation of the monoenoic fatty acids depending on the ethylenic double bond (C═C) configuration.

Selective deletion of the internal lysine residue from the peptide sequence by collisional activation.

The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment.

Electrospray ionization mass spectrometry: a technique to access the information beyond the molecular weight of the analyte.

The Electrospray Ionization (ESI) is a soft ionization technique extensively used for production of gas phase ions (without fragmentation) of thermally labile large supramolecules. In the present review we have described the development of Electrospray Ionization mass spectrometry (ESI-MS) during the last 25 years in the study of various properties of different types of biological molecules. There have been extensive studies on the mechanism of formation of charged gaseous species by the ESI.

Sequence specific association of tryptic peptides with multiwalled carbon nanotubes: effect of localization of hydrophobic residues.

A model-free approach has been used to study the association of peptides onto multiwalled carbon nanotubes (MWCNT) in aqueous solution at ambient pH to understand the molecular basis of interaction of the peptides with MWCNT. The peptides obtained by tryptic digestion of cytochrome P450cam from P. putida were allowed to interact with MWCNT, and several peptides were found to bind to the nanotube leading to formation of stable homogeneous dispersion of the bionano conjugates of MWCNT.