Dr. Amit Kumar

University at Buffalo, The State University of New York | SUNY Buffalo · Department of Biochemistry

Myoglobin (Mb) uses strong electrostatic interaction in its distal heme pocket to regulate ligand binding. The mechanism of regulation of ligand binding in soybean leghemoglobin a (Lba) has been enigmatic and more so due to the absence of gaseous ligand bound atomic resolution three-dimensional structure of the plant globin. While the 20-fold highe...

A novel heme-peroxidase has been isolated from the medicinally important plant Artocarpus lakoocha and preliminary studies suggest it has anti-inflammatory and wound healing properties. The protein's structure, dynamics, and stability have been examined using Far-UV Circular Dichroism, fluorescence spectroscopic studies, and activity measurements....

A series of imidazole and triazole diarylpyrazole derivatives were prepared using an efficient 5-step synthetic scheme and evaluated for binding affinity with Mycobacterium tuberculosis (Mtb) CYP121A1 and antimycobacterial activity against Mtb H37Rv. Antimycobacterial susceptibility was measured using the spot-culture growth inhibition assay (SPOTi...

The complex enzyme kinetics displayed by drug-metabolizing cytochrome P450 enzymes (CYPs) (see Chapter 9) can, in part, be explained by an examination of their crystallographic protein structures. Fortunately, despite low sequence similarity between different families of drug-metabolizing CYPs, there exists a high degree of structural homology with...

The mitochondrial cytochrome P450 24A1 (CYP24A1) is necessary for the deactivation of bioactive vitamin-D (1,25(OH)2D3) by side-chain hydroxylation. The enzyme requires molecular recognition by its cognate redox partner adrenodoxin (Adx), which houses an iron-sulfur cluster and provides two electrons for the hydroxylation step to achieve the reduct...

Tuberculosis is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) and remains the leading cause of death by infection world-wide. Out of twenty cytochrome P450 enzymes encoded by the Mtb genome, cytochrome P450 121A1 (CYP121A1) is essential, and therefore remains a target of drug design efforts. CYP121A1 mediates a phenol coupling...

Tuberculosis is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) and remains the leading cause of death by infection world-wide. The Mtb genome encodes a disproportionate number of twenty cytochrome P450 enzymes, of which the essential enzyme cytochrome P450 121A1 (CYP121A1) remains a target of drug design efforts. CYP121A1 media...

Metabolic deactivation of 1,25(OH)2D3 is initiated by modification of the vitamin-D side chain, as carried out by the mitochondrial cytochrome P450 24A1 (CYP24A1). In addition to its role in vitamin-D metabolism, CYP24A1 is involved in catabolism of vitamin-D analogs, thereby reducing their efficacy. CYP24A1 function relies on electron transfer fro...

During the adaptive evolution of a particular trait, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation to adaptive phenotypic evolution depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitu...

Stability of α-helical secondary structure as a function of pH, measured by circular dichroism spectroscopy. Measurements of molar ellipticity are shown for rHbs representing wildtype genotypes of bar-headed goose (BHG), greylag goose (GG), their reconstructed ancestor (AncAnser), and all possible mutational intermediates connecting AncAnser with e...

The αP119A mutation has consistent effects on Hb-O2 affinity on different genetic backgrounds. The affinity-enhancing effect of αP119A on the AncAnser background is mirrored by a similarly pronounced affinity-reducing effect when the mutation is reverted on the wildtype bar-headed goose background (αA119P). By contrast, forward and reverse mutation...

Variation among goose rHb mutants in functional and structural properties that potentially trade-off with intrinsic O2 affinity. Variation in (A) autoxidation rate (rate at which ferrous heme [Fe2+] spontaneously oxidizes to the ferric state [Fe3+]), (B) secondary structure content, as assessed by means of circular dichroism spectra (with elliptici...

Ramachandran plot of deoxyHb from bar-headed goose (PDB 1hv4). Glycine residues are denoted by triangles, other residues by squares. One residue, α18-Ser, is conspicuous by its unusual backbone angles, and is shown as a green square. This position in the Ramachandran plot is highly unusual for any residue other than glycine. The turn in the backbon...

Effect of pH on the stability of tertiary structure, as measured by UV-visible spectroscopy. Measurements of absorbance maxima at 412 nm are shown for rHbs representing wildtype genotypes of bar-headed goose (BHG), greylag goose (GG), their reconstructed ancestor (AncAnser), and all possible mutational intermediates connecting AncAnser with each of...

A comparative study of tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a in the oxy- and deoxy-forms was carried out using (57)Fe Mössbauer spectroscopy with a high velocity resolution in order to analyze the heme iron electronic structure and stereochemistry in relation to the Mössbauer hyperfine parameters. The Mössbauer spectra...

During adaptive phenotypic evolution, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitutions and their pleiotropic effects, we used a p...

If the fitness effects of amino acid mutations are conditional on genetic background, then mutations can have different effects depending on the sequential order in which they occur during evolutionary transitions in protein function. A key question concerns the fraction of possible mutational pathways connecting alternative functional states that...

Hemoglobins with diverse characteristics have been identified in all kingdoms of life. Their ubiquitous presence indicates that these proteins play important roles in physiology, though function for all hemoglobins are not yet established with certainty. Their physiological role may depend on their ability to bind ligands, which in turn is dictated...

The 57Fe hyperfine interactions are the useful source of information about the finest structural peculiarities in various iron-containing proteins which can be essential in the natural variety of structure and function relationship in normal biomolecules as well as in its changes in the case of molecular pathology. Mössbauer spectroscopy with a hig...

The bar-headed goose (‘BHG’, Anser indicus) is renowned for its trans-Himalayan migratory flights, and the elevated hemoglobin (Hb)-O2 affinity of this species is thought to make a key contribution to its capacity for powered flight at elevations of ~9000 m. Here we revisit the molecular basis of this text-book example of biochemical adaptation. Pr...

An analysis of the Mössbauer spectra of soybean leghemoglobin a in both oxy and deoxyforms measured with a high velocity resolution at 90 K was carried out in comparison with the room temperature Mössbauer spectrum of the standard absorber sodium nitroprusside. On the basis of the observed features of the spectral line shapes the soybean leghemoglo...

Artocarpus lakoocha is a medicinal plant used in cosmetics and traditional remedies of many diseases. We have purified a protein from its latex using anion exchange chromatography and gel filtration, which resulted in 28.82% yield of protein, with specific activity of 10.8 units/mg. FPLC spectroscopy and isoelectrofocusing demonstrated that purifie...

A comparative study of monomeric soybean and lupin leghemoglobins in the oxy-form was carried out using Mössbauer spectroscopy with a high velocity resolution at 90 K. The 57Fe hyperfine parameters of measured spectra were evaluated and compared with possible structural differences in the heme Fe(II)-O 2 bond.

The ubiquity of hemoglobins as a superfamily to life has enthused the field with renewed vigor. Reactions like oxygen binding and nitric oxide (NO) dioxygenation appear to be characteristic to the hemoglobin superfamily, as revealed from investigation of recombinant globins, irrespective of whether they are associated to any particular function lik...

A preliminary investigation of monomeric soybean leghemoglobin a in oxy-and deoxy-forms was carried out for the first time using Mössbauer spectroscopy with a high velocity resolution at 90 K. Mössbauer parameters of measured spectra were evaluated and compared with that for the results of the well-known monomeric heme protein, myoglobin.

Atrazine is a selective herbicide used in agricultural fields to control the emergence of broadleaf and grassy weeds. The persistence of this herbicide is influenced by the metabolic action of habituated native microorganisms. This study provides information on the occurrence of atrazine mineralizing bacterial strains with faster metabolizing abili...

Normal human adult, rabbit and pig oxyhemoglobins in frozen red blood cell solutions were studied using Mössbauer spectroscopy with a high velocity resolution. Spectra were analyzed using two models with and without accounting for the heme iron electronic structure non-equivalence in α- and β-subunits of tetrameric hemoglobins. The observed differe...

A comparative study of oxyhemoglobins from pig, rabbit, normal human and patients with blood system malignant diseases was performed using Mössbauer spectroscopy with a high velocity resolution at 90 K. Mössbauer spectra were fitted with the help of two models: using one quadrupole doublet (model of equivalent iron electronic structure in α- and β-...

A comparative study of normal human, rabbit and pig oxyhemoglobins and oxyhemoglobin from patients with chronic myeloleukemia and multiple myeloma using Mössbauer spectroscopy with a high velocity resolution demonstrated small variations of the 57Fe quadrupole splitting and isomer shift. These variations may be a result of small structural differen...

Comparison of the heme iron electronic structure in various hemoglobins is very important in order to analyze structure-function relationship of oxygen carriers. It is well known that small stereochemical differences exist in the heme iron in different hemoglobins as well as in non-identical subunits in tetramer. Mössbauer spectroscopy is the most...