Effects of Temperature on the Kinetic Isotope Effects for Proton and Hydride Transfers in the Active Site Variant of Choline Oxidase Ser101Ala

Choline oxidase catalyzes the oxidation of choline to glycine betaine. The reaction includes betaine aldehyde as an intermediate. FAD is reduced by the alcohol substrate, betaine aldehyde intermediate and oxidized by molecular oxygen to give hydrogen peroxide. In this study, the Ser101Ala variant of choline oxidase was prepared to elucidate the contribution of the hydroxyl group of Ser101 in the proton and hydride transfer reactions for proper preorganization and reorganization of the active site towards quantum mechanical tunneling. The thermodynamic parameters associated with the enzyme-catalyzed OH and CH bond cleavages and the temperature dependence of the associated solvent and substrate kinetic isotope effects were investigated using a stopped-flow spectrophotometer. The proton and hydride transfer have been shown to be occurring via quantum tunneling in CHO-S101A enzyme

Biochemical and Biophysical Studies of Heme Transport Proteins: HtaA, HtaB, and ChtB from the Corynebacterium diphtheriae

Many pathogenic bacteria require iron for their survival and virulence; in most cases hemin is the main iron source. Pathogens have developed sophisticated heme uptake mechanisms in order to maintain the homeostasis and remain as infectious agents. Corynebacterium diphtheria can obtain hemin during human infection through series of conserved domains (CR) of DxtR-regulated and heme-transport-associated (hta) proteins: HtaA, HtaB, ChtA, ChtB and ChtC. HtaA includes two conserved regions (CR1 and CR2) while the other proteins include a single CR domain. These proteins orchestrate the heme transport to the HmuT protein, a lipoprotein which delivers heme to the ABC membrane transporter HmuUV. Homology modeling of HtaA-CR2, HtaB and ChtB based on amino acid sequence indicated that these proteins have a novel structure. Two tyrosines and one histidine residue are fully conserved in all CR domains. Mutations of these conserved amino acids to alanine significantly lowered the heme binding in comparison to the wild-type proteins. Reconstitution of HtaA-CR2 after removal of the heme with butanone extraction method gave a different form of the protein. UV-visible absorption spectra and resonance Raman spectra data are consistent with heme ligation with an axial tyrosine including a histidine hydrogen-binding partner in HtaA-CR2, HtaB and ChtB. HtaA-CR2 is highly stable to thermal unfolding; the protein was also stable to chemical unfolding using GdnHCl or GdnSCN (up to 4 M at 25oC) . For HtaA-CR2, unfolding could be observed at 37 oC as a single process at high concentrations of denaturant (6.8 – 7.4 M GdnHCl). In contrast, HtaA-CR2 apoproteins (WT, as well as the Y361A and H412A mutants) unfolded readily with low denaturant concentrations (~ 1.3 M GdnHCl). HtaB shows significantly lower stability; a half-life of 330 min was observed in the presence of 6.6 M GdnHCl at 37 oC for HtaA-CR2 and a half-life of 39 min was observed in the presence of 4.0 M GdnHCl at 25 oC for HtaB; ChtB was very similar to HtaB. For HtaB and ChtB, the high amino acid sequence similarity and identity, similar biophysical characteristics and gene deletion studies suggest that these proteins may function interchangeably during the heme uptake process

Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance

Genetic improvement of tomato by targeted control of fruit softening

Controlling the rate of softening to extend shelf life was a key target for researchers engineering genetically modified (GM) tomatoes in the 1990s, but only modest improvements were achieved. Hybrids grown nowadays contain 'non-ripening mutations' that slow ripening and improve shelf life, but adversely affect flavor and color. We report substantial, targeted control of tomato softening, without affecting other aspects of ripening, by silencing a gene encoding a pectate lyase

Studies of HtaB from Corynebacterium diphtheriae

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