Novel B(12)-Dependent Acyl-CoA Mutases and Their Biotechnological Potential

The recent spate of discoveries of novel acyl-CoA mutases has engendered a growing appreciation for the diversity of 5'-deoxyadenosylcobalamin-dependent rearrangement reactions. The prototype of the reaction catalyzed by these enzymes is the 1,2 interchange of a hydrogen atom with a thioester group leading to a change in the degree of carbon skeleton branching. These enzymes are predicted to share common architectural elements: a Rossman fold and a triose phosphate isomerase (TIM)-barrel domain for binding cofactor and substrate, respectively. Within this family, methylmalonyl-CoA mutase (MCM) is the best studied and is the only member found in organisms ranging from bacteria to man. MCM interconverts (2R)-methylmalonyl-CoA and succinyl-CoA. The more recently discovered family members include isobutyryl-CoA mutase (ICM), which interconverts isobutyryl-CoA and n-butyryl-CoA; ethylmalonyl-CoA mutase, which interconverts (2R)-ethylmalonyl-CoA and (2S)-methylsuccinyl-CoA; and 2-hydroxyisobutyryl-CoA mutase, which interconverts 2-hydroxyisobutyryl-CoA and (3S)-hydroxybutyryl-CoA. A variant in which the two subunits of ICM are fused to a G-protein chaperone, IcmF, has been described recently. In addition to its ICM activity, IcmF also catalyzes the interconversion of isovaleryl-CoA and pivalyl-CoA. This review focuses on the involvement of acyl-CoA mutases in central carbon and secondary bacterial metabolism and on their biotechnological potential for applications ranging from bioremediation to stereospecific synthesis of C2-C5 carboxylic acids and alcohols, and for production of potential commodity and specialty chemicals.     

Dioxygen reactivity and heme redox potential of truncated human cystathionine beta-synthase

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 +/- 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 +/- 0.07) x 10 (5) M (-1) s (-1) at pH 7.4 and 25 degrees C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical.     

Redox Biochemistry of Hydrogen Sulfide

H₂S, the most recently discovered gasotransmitter, might in fact be the evolutionary matriarch of this family, being both ancient and highly reduced. Disruption of γ-cystathionase in mice leads to cardiovascular dysfunction and marked hypertension, suggesting a key role for this enzyme in H₂S production in the vasculature. However, patients with inherited deficiency in γ-cystathionase apparently do not present vascular pathology. A mitochondrial pathway disposes sulfide and couples it to oxidative phosphorylation while also exposing cytochrome c oxidase to this metabolic poison. This report focuses on the biochemistry of H₂S biogenesis and clearance, on the molecular mechanisms of its action, and on its varied biological effects.     

Phenethyl isothiocyanate,by virtue of its antioxidant activity,inhibits invasiveness and metastatic potential of breast cancer cells: HIF-1α as a putative target

Hypoxia-inducible factor 1α (HIF-1α) plays a crucial role in facilitating tumor progression and metastasis. Reducing the levels of HIF-1α might therefore be an important anticancer strategy. This could be achieved by understanding the key cellular events involved in HIF-1α activation. Present study explored the effect of phenethyl isothiocyanate (PEITC), a natural isothiocyanate, found in cruciferous vegetables on the expression of HIF-1α and HSP90 in breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) under both normoxia and hypoxia. This study established the possible role of ROS in the up-regulation of these markers in breast cancer cells. PEITC-induced nuclear accumulation of Nrf2, increased the activities of several antioxidant enzymes, and thus reduced the ROS burden of the tumor cells by acting as an indirect antioxidant. This resulted in the down-regulation of HSP90 and thereby HIF-1α expression. HSP90 was also found to be involved in the regulation of HIF-1α. A probable link between down-regulation of HIF-1α with reduction of ROS by PEITC through induction of Nrf2 was determined. Finally, our study demonstrated that modulation of HIF-1α by PEITC retarded adhesion, aggregation, migration and invasion of the breast cancer cells, thereby showing anti-metastatic effect. Activities of MMPs (2 & 9) and expression of VEGF were also altered by PEITC.     

Magnetic field effects on coenzyme B12-dependent enzymes: Validation of ethanolamine ammonia lyase results and extension to human methylmalonyl CoA mutase

Enzymes with radical-pair intermediates have been considered as a likely target for purported magnetic field effects in humans. The bacterial enzyme ethanolamine ammonia lyase and the human enzyme methylmalonyl-CoA mutase catalyze coenzyme B₁₂-dependent rearrangement reactions. A common step in the mechanism of these two enzymes is postulated to be homolysis of the cobalt-carbon bond of the cofactor to generate a spin-correlated radical pair consisting of the 5′-deoxyadenosyl radical and cob(II)alamin [Ado^· Cbl(II)]. Thus, the reactions catalyzed by these enzymes are expected to be sensitive to an applied magnetic field according to the same principles that control radical pair chemical reactions. The magnetic field effect on ethanolamine ammonia lyase reported previously has been corroborated independently in one of the authors' laboratory. However, neither the human nor the bacterial mutase from Propionibacterium shermanii exhibits a magnetic field effect that could be greater than about 15%, considering the error limit imposed by the uncertainty of the coupled assay. Our studies suggest that putative magnetic field effects on physiological processes are not likely to be mediated by methylmalonyl-CoA mutase. Bioelectromagnetics 18:506–513, 1997. © 1997 Wiley-Liss, Inc.     

DNAX-activating Protein 10 (DAP10) Membrane Adaptor Associates with Receptor for Advanced Glycation End Products (RAGE) and Modulates the RAGE-triggered Signaling Pathway in Human Keratinocytes

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.     

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO^•), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O₂ to Fe(III)-CBS, forming superoxide radical anion (O₂^˙̄). In this study, we describe the kinetics of nitrite (NO₂⁻) reduction by Fe(II)-CBS to form Fe(II)NO^•-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO^•-CBS by O₂ showed complex kinetic behavior and led to peroxynitrite (ONOO⁻) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO^• and peroxynitrite.     

Phytoplankton and nutrient dynamics of shallow coastal stations at Bay of Bengal,Eastern Indian coast

Phytoplankton dynamics of Eastern Indian coast was studied from surface water for a period of 24 months (April 2005–March 2007) in relation to environmental variables like, temperature, pH, dissolved oxygen, biochemical oxygen demand (BOD), salinity and nutrient contents—including nitrate, phosphate and silicate. Total 43 taxa were recorded during the study period. Phytoplankton density ranged from approximately 350–3,000 cells/ml and showed complete dominance of diatom genera namely, Asterionella japonica in winter and Odontella rhombus in summer. Other frequently occurring diatoms were Coscinodiscus perforatus, Actinocyclus normanii f. subsala, Thalassiothrix fraunfeldii, Ditylum brightwelli, Stephanodiscus hantzschoides, Cyclotella meneghiniana, Thalassionema nitzschoides etc. Seasonal changes in abundance and diversity of phytoplankton significantly differed showing maximum diversity in autumn with high diversity index (2.76-Oct, 05) and minimum in winter (0.326-January, 06). The species evenness varied from 0.137 (January 06) to 0.991 (August 06), which signifies minimum variation in percentage contribution of individual species to total phytoplankton population in monsoon and maximum in winter. Correlation studies of total cell count to physicochemical variables indicated significant positive relation with dissolved oxygen, salinity and pH but negative relation with nitrate, silicate and BOD of the water body. Multivariate procedures like ordination by principal component analysis and multi dimensional scaling of phytoplankton population based on their occurrence data and magnitude of abundance indicated that some genera (Biddulphia heteroceros, B. dubia, Odontella aurita, Gyrosigma acuminatum, Coscinodiscus granii, Paralia sulcata, etc.) have specific preference for water temperature and salinity and flourished maximally in particular season(s). While other genera (A. japonica, C. meneghiniana, C. perforatus, D. brightwelli, S. hantzschoides, etc.) appeared in wide range of temperature and salinity gradient.