Structure-function relationship in serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5V-phosphate (PLP)-dependent enzyme catalyzes thetetrahydrofolate (H4-folate)- dependent retro-aldol cleavage of serine to form 5,10-methylene H4-folate and glycine. The structure–function relationship of SHMT wasstudied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site. The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure. The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis. E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an ‘open’ to ‘closed’ conformation rather than proton abstraction from the hydroxylgroup of serine. K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure. Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate.The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s)complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT.

A touch of history and a peep into the future of the lipid-quinone known as coenzyme Q and ubiquinone

Coenzyme Q (ubiquinone), a fully substituted benzoquinone with polyprenyl side chain, participates in many cellular redox activities. Paradoxically it was discovered only in 1957, albeit being ubiquitous. It required a person, F. L. Crane, a place, Enzyme Institute, Madison, USA, and a time when D. E. Green was directing vigorous research on mitochondria. Located at the transition of 2-electron flavoproteins and 1-electron cytochrome carriers, it facilitates electron transfer through the elegant Q-cycle in mitochondria to reduce O-2 to H2O, and to H2O2, now a significant signal-transducing agent, as a minor activity in shunt pathway (animals) and alternative oxidase (plants). The ability to form Q-radical by losing an electron and a proton was ingeniously used by Mitchell to explain the formation of the proton gradient, considered the core of energy transduction, and also in acidification in vacuoles. Known to be a mobile membrane constituent (microsomes, plasma membrane and Golgi apparatus), allowing it to reach multiple sites, coenzyme Q is expected to have other activities. Coenzyme Q protects circulating lipoproteins being a better lipid antioxidant than even vitamin E. Binding to proteins such as QPS, QPN, QPC and uncoupling protein in mitochondria, QA and QB in the reaction centre in R. sphaeroides, and disulfide bond-forming protein in E. coli (possibly also in Golgi), coenzyme Q acquires selective functions. A characteristic of orally dosed coenzyme Q is its exclusive absorption into the liver, but not the other tissues. This enrichment of Q is accompanied by significant decrease of blood pressure and of serum cholesterol. Inhibition of formation of mevalonate, the common precursor in the branched isoprene pathway, by the minor product, coenzyme Q, decreases the major product, cholesterol. Relaxation of contracted arterial smooth muscle by a side-chain truncated product of coenzyme Q explains its effect of decreasing blood pressure. Extensive clinical studies carried out on oral supplements of coenzyine Q, initially by K. Folkers and Y. Yamamura and followed many others, revealed a large number of beneficial effects, significantly in cardiovascular diseases. Such a variety of effects by this lipid quinone cannot depend on redox activity alone. The fat-soluble vitamins (A, D, E and K) that bear structural relationship with coenzyme Q are known to be active in their polar forms. A vignette of modified forms of coenzyme Q taking active role in its multiple effects is emerging.

Biotin-conjugated tumour-targeting photocytotoxic iron(III) complexes

Iron(III) complexes FeL(B)] (1-4) of a tetradentate phenolate-based ligand (H3L) and biotin-conjugated dipyridophenazine bases (B), viz. 7-aminodipyrido 3,2-a: 2',3'-c]-phenazine (dppza in 1), (N-dipyrido3,2-a: 2',3'-c]-phenazino) amidobiotin (dppzNB in 2), dipyrido 3,2-a: 2',3'-c]-phenazine-11-carboxylic acid (dppzc in 3) and 2-((2-biotinamido) ethyl) amidodipyrido 3,2-a: 2',3'-c]-phenazine (dppzCB in 4) are prepared, characterized and their interaction with streptavidin and DNA and their photocytotoxicity and cellular uptake in various cells studied. The high-spin iron(III) complexes display Fe(III)/Fe(II) redox couple near -0.7V versus saturated calomel electrode in dimethyl sulfoxide-0.1M tetrabutylammonium perchlorate. The complexes show non-specific interaction with DNA as determined from the binding studies. Complexes with appended biotin moiety show similar binding to streptavidin as that of free biotin, suggesting biotin conjugation to dppz does not cause any loss in its binding affinity to streptavidin. The photocytotoxicity of the complexes is tested in HepG2, HeLa and HEK293 cell lines. Complex 2 shows higher photocytotoxicity in HepG2 cells than in HeLa or HEK293, forming reactive oxygen species. This effect is attributed to the presence of overexpressed sodium-dependent multi-vitamin transporters in HepG2 cells. Microscopic studies in HepG2 cells show internalization of the biotin complexes 2 and 4 essentially occurring by receptor-mediated endocytosis, which is similar to that of native biotin and biotin fluorescein isothiocyanate conjugate.

Rapid determination of main constituents of packed juices by reverse phase-high performance liquid chromatography: an insight in to commercial fruit drinks

The present work reports the compositional analysis of thirteen different packed fruit juices using high performance liquid chromatography (HPLC). Vitamin C, organic acids (citric and malic) and sugars (fructose, glucose and sucrose) were separated, analyzed and quantified using different reverse phase methods. A new rapid reverse phase HPLC method was developed for routine analysis of vitamin C in fruit juices. The precision results of the methods showed that the relative standard deviations of the repeatability and reproducibility were < 0.05 and < 0.1 respectively. Correlation coefficient of the calibration models developed was found to be higher than 0.99 in each case. It has been found that the content of Vitamin C was less variable amongst different varieties involved in the study. It is also observed that in comparison to fresh juices, the packed juices contain lesser amounts of vitamin C. Citric acid was found as the major organic acids present in packed juices while maximum portion of sugars was of sucrose. Comparison of the amount of vitamin C, organic acids and sugars in same fruit juice of different commercial brands is also reported.

Successful data recovery from oscillation photographs containing strong polycrystalline diffraction rings from an unknown small-molecule contaminant: preliminary structure solution of Salmonella typhimurium pyridoxal kinase (PdxK)

Pyridoxal kinase (PdxK; EC 2.7.1.35) belongs to the phosphotransferase family of enzymes and catalyzes the conversion of the three active forms of vitamin B-6, pyridoxine, pyridoxal and pyridoxamine, to their phosphorylated forms and thereby plays a key role in pyridoxal 5 `-phosphate salvage. In the present study, pyridoxal kinase from Salmonella typhimurium was cloned and overexpressed in Escherichia coli, purified using Ni-NTA affinity chromatography and crystallized. X-ray diffraction data were collected to 2.6 angstrom resolution at 100 K. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unitcell parameters a = 65.11, b = 72.89, c = 107.52 angstrom. The data quality obtained by routine processing was poor owing to the presence of strong diffraction rings caused by a polycrystalline material of an unknown small molecule in all oscillation images. Excluding the reflections close to powder/polycrystalline rings provided data of sufficient quality for structure determination. A preliminary structure solution has been obtained by molecular replacement with the Phaser program in the CCP4 suite using E. coli pyridoxal kinase (PDB entry 2ddm) as the phasing model. Further refinement and analysis of the structure are likely to provide valuable insights into catalysis by pyridoxal kinases.

Iron(III) Complexes of a Pyridoxal Schiff Base for Enhanced Cellular Uptake with Selectivity and Remarkable Photocytotoxicity

Iron(III) complexes of pyridoxal (vitamin B6, VB6) or salicylaldehyde Schiff bases and modified dipicolylamines, namely, Fe(B)(L)](NO3) (15), where B is phenyl-N,N-bis((pyridin-2-yl)methyl)methanamine (phbpa in 1), (anthracen-9-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (anbpa in 2, 4) and (pyren-1-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (pybpa in 3, 5) (H2L1 is 3-hydroxy-5-(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-methylp yridine (13) and H2L2 is 2-(2-hydroxyphenyl-imino)methyl]phenol), were prepared and their uptake in cancer cells and photocytotoxicity were studied. Complexes 4 and 5, having a non-pyridoxal Schiff base, were prepared to probe the role of the pyridoxal group in tumor targeting and cellular uptake. The PF6 salt (1a) of complex 1 is structurally characterized. The complexes have a distorted six-coordinate FeN4O2 core where the metal is in the +3 oxidation state with five unpaired electrons. The complexes display a ligand to metal charge transfer band near 520 and 420 nm from phenolate to the iron(III) center. The photophysical properties of the complexes are explained from the time dependent density functional theory calculations. The redox active complexes show a quasi-reversible Fe(III)/Fe(II) response near -0.3 V vs saturated calomel electrode. Complexes 2 and 3 exhibit remarkable photocytotoxicity in various cancer cells with IC50 values ranging from 0.4 to 5 mu M with 10-fold lower dark toxicity. The cell death proceeded by the apoptotic pathway due to generation of reactive oxygen species upon light exposure. The nonvitamin complexes 4 and 5 display 3-fold lower photocytotoxicity compared to their VB6 analogues, possibly due to preferential and faster uptake of the vitamin complexes in the cancer cells. Complexes 2 and 3 show significant uptake in the endoplasmic reticulum, while complexes 4 and 5 are distributed throughout the cells without any specific localization pattern.

Simultaneous Determination of Catecholamines in Presence of Uric Acid and Ascorbic Acid at a Highly Sensitive Electrochemically Activated Carbon Paste Electrode

A simple yet remarkable, electrochemically activated carbon paste electrode (EACPE) was prepared by successive potential cycling of carbon paste in a 0.1 M NaOH solution and was effectively used for the simultaneous determination of catecholamines such as dopamine (DA), epinephrine (E) and Norepinephrine (NE) in presence of uric acid (UA) and ascorbic acid (AA). Taking DA as the ideal catecholamine, the electrochemical behaviors of DA, UA and AA such as scan rate and pH variation was studied by cyclic voltammetry (CV) in phosphate buffer solution (PBS, pH 7.1). This electrochemical sensor exhibited strong electrocatalytic activity towards the oxidation of a mixture of catecholamines, UA and AA with apparent reduction of overpotentials. Crider optimum conditions, limit of detection (S/N = 3) of DA, E, NE, UA and AA was found to be 0.08, 0.08, 0.07, 0.1 and 6.0 mu M, respectively by differential pulse voltammetry (DPV). The analytical performance of this modified electrode as a biosensor was also demonstrated for the determination of DA, UA and AA in dopamine injection, human urine and vitamin C tablets, respectively, in presence of other interfering substances. (C) 2015 The Electrochemical Society. All-rights reserved.

Multicomponent Adducts of Pyridoxine: An Evaluation of the Formation of Eutectics and Molecular Salts

Cocrystallization of pyridoxine (vitamin B6) with several biologically important molecules was undertaken with the intent of successfully designing various hydrogen bonded adducts such as salts, cocrystals, and eutectics. Pyridoxine formed eutectics with isoniazid (an antitubercular drug) and nicotinic acid (vitamin B3) and molecular salts with para-aminobenzoic acid (a bioactive) and saccharin (an artificial sweetener), respectively, in accordance to our design strategy. A salt cocrystal, a precisely conjugate acid-base cocrystal, was obtained for the pyridoxine-para-nitrobenzoic acid combination. The role of supramolecular affinity of hydrogen bonding functional groups and Delta pK(a) differences leading to the formation of above diverse adducts was discussed. This study underpins the need for full-fledged supramolecular compatibility studies of multivitamin/drug combinations toward the development of optimal and/or synergistic combination formulations.

The putative role of some conserved water molecules in the structure and function of human transthyretin

Human transthyretin (hTTR) is a multifunctional protein that is involved in several neurodegenerative diseases. Besides the transportation of thyroxin and vitamin A, it is also involved in the proteolysis of apolipoprotein A1 and A beta peptide. Extensive analyses of 32 high-resolution X-ray and neutron diffraction structures of hTTR followed by molecular-dynamics simulation studies using a set of 15 selected structures affirmed the presence of 44 conserved water molecules in its dimeric structure. They are found to play several important roles in the structure and function of the protein. Eight water molecules stabilize the dimeric structure through an extensive hydrogen-bonding network. The absence of some of these water molecules in highly acidic conditions (pH <= 4.0) severely affects the interfacial hydrogen-bond network, which may destabilize the native tetrameric structure, leading to its dissociation. Three pairs of conserved water molecules contribute to maintaining the geometry of the ligand-binding cavities. Some other water molecules control the orientation and dynamics of different structural elements of hTTR. This systematic study of the location, absence, networking and interactions of the conserved water molecules may shed some light on various structural and functional aspects of the protein. The present study may also provide some rational clues about the conserved water-mediated architecture and stability of hTTR.

Electron transfer amongst flavo- and hemo-proteins: diffusible species effect the relay processes, not protein-protein binding

Hitherto, electron transfer (ET) between redox proteins has been deemed to occur via donor-acceptor binding, and diffusible reactive species are considered as deleterious side-products in such systems. Herein, ET from cytochrome P450 reductase (CPR, an animal membrane flavoprotein) and horseradish peroxidase (HRP, a plant hemoprotein) to cytochrome c (Cyt c, a soluble animal hemoprotein) was probed under diverse conditions, using standard assays. ET in the CPR-Cyt c system was critically inhibited by cyanide and sub-equivalent levels of polar one-electron cyclers like copper ions, vitamin C/Trolox and superoxide dismutase. In the presence of lipids, inhibition was also afforded by amphipathic molecules vitamin E, palmitoyl-vitamin C and the membrane hemoprotein, cytochrome b(5). Such nonspecific inhibition (by diverse agents in both aqueous and lipid phases) indicated that electron transfer/relay was effected by small diffusible agents, whose lifetimes are shortened by the diverse radical scavengers. When CPR was retained in a dialysis membrane and Cyt c presented outside in free solution, ET was still observed. Further, HRP (taken at nM levels) catalyzed oxidation of a phenolic substrate was significantly inhibited upon the incorporation of sub-nM levels of Cyt c. The findings imply that CPR-Cyt c or HRP-Cyt c binding is not crucial for ET. Further, fundamental quantitative arguments (based on diffusion/collision) challenge the erstwhile protein-protein binding-assisted ET hypothesis. It is proven beyond reasonable doubt that mobile and diffusible electron carriers (ions and radicals) serve as ``redox-relay agents'' in the biological ET models/setup studied.