Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus

Background: Glycine oxidase (GO), a type of D-amino acid oxidase, is of biotechnological interest for its potential in several fields. In our previous study, we have characterized a new glycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against all the tested substrate was obtained by screening a random mutant library of BceGO. It is observed that the residue N336 is invariable between its homogeneous enzymes. This work was aimed to explore the role of the residue N336 in glycine oxidase by site-directed mutagenesis, kinetic assay, structure modeling and substrate docking. Results: The results showed that the affinity of N336H, N336K and N336R increased gradually toward all the substrates, with increase in positive charge on side chain, while N336A and N336G have not shown a little significant effect on substrate affinity. The structure modeling studies indicated that the residue Asn336 is located in a random coil between -J-18 and a-10. Also, far-UV CD spectra-analysis showed that the mutations at Asn336 do not affect the secondary structure of enzyme. Conclusion: Asn336 site was located in a conserved GHYRNG loop which adjoining to substrate and the isoalloxazine ring of FAD, and involved in the substrate affinity of glycine oxidase. This might provide new insight into the structure-function relationship of GO, and valuable clue to redesign its substrate specificity for some biotechnological application.

Isolation, purification, and characterization of L-glutamate oxidase from Streptomyces sp. 18G

An extracellular L-glutamate oxidase (GLOD) was purified from soil-isolated Streptomyces sp 18G. The enzyme had a molecular weight of approximately 120,000 and consisted of two identical subunits, each with a molecular weight of 61,000. The isoelectric point was pH 8.5 and the enzyme had an optimal pH between 7.0-7.4. GLOD showed the maximum activity at 37ºC. The GLOD activity was stable at pH ranging from 6.5 to 7.0 for 1 hr. Among 21 amino acids tested for substrate specificity, L-glutamate was almost exclusively oxidized. D-glutamate and L-aspartate were oxidized but only to extents of 0.79 percent and 0.53 percent, respectively.

Enzymes that regulate ethylene levels--1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, ACC synthase and ACC oxidase.

The plant enzymes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase, catalyze essential steps in the biosynthesis of the phytohormone ethylene; the microbial enzyme ACC deaminase catalyses the hydrolytic cleavage of ACC, the immediate precursor of ethylene, and is therefore an inhibitor of ethylene biosynthesis. In this manuscript, the biochemical properties and mechanisms of these three enzymes and the genes that encode them are examined and compared. Despite the fact that ACC oxidase and ACC deaminase both act on the same substrate, i.e., ACC, these two enzymes and the mechanisms that they employ are quite different. Conversely, although ACC synthase catalyses the synthesis of ACC and ACC deaminase catalyses its hydrolysis, these enzymes share a number of important physical and biochemical properties.

Biochemical characterization of NADPH diaphorase in the chick embryo retina: stimulation by calcium ions and inhibition by arginine analogs

Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is prsent in specific groups of cells in several neural tissues, including the retina. We determined NADPH-diaphorase activity in homogenates of the chick embryo retina. The enzyme activity was measured spectophotometrically at 585 nm after incubating retinal total homogenates (100-150 µg protein) with 1mMNADPH and 0.5 mM nitroblue tetrazolium in 50 mMTris buffer, pH8.1, at 37ºC. NADPH-diaphorse was detected in 14-day old retinas and 53-65 per cent of the enzyme activity was inhibited by 3 mM NG-nitro-L-arginine (NARG), the arginine analog. One mM L-N5-(1-iminoethyl)ornithine (NIO) was the most potent inhibitor (63 per cent inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33 per cent inhibition) and I mMNG-monomethyl-L-arginine acetate (NMMA) (14 per cent inhbition) were less effective. Enzyme activity was increased by 48 per cent by 2 mM calcium chloride, and effect reversed by 1 mMEGTA or EDTA. Basal enzyme levels were also partially inhibited by the chelators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show that the NADPH-diaphorase assay is sample and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated

Negative inotropic effect of carbachol on rat atria mediated by nitric oxide

En este trabajo nosotros mostramos que la aurícula aislada de rata sintetiza óxido nítrico (ON) y este actúa como un mensajero intracelular incrementando la producción de GMPc, que a su vez modula el efecto contráctil inhibitorio ejercido por la activación muscarínica. El carbacol activando al receptor muscrínico M2 estimula el ciclo de los fosfoinosítidos y a la óxido nítrico sintaza con la consiguiente producción de ON. Los inhibidores de fosfolipasa C, proteína quinasa C, calcio/calmodulina, óxido nítrico sintaza y guanilato ciclasa, desvían hacia la derecha la curva dosis-respuesta del carbacol sobre la contractilidad. Más aun, en nitroprusiato de sodio y el 8-bromo GMPc indujeron un efecto inotrópico negativo, similar a las bajas concentraciones de carbacol. Estos reultados sugieren que el carbacol activando a receptores muscarínicos de tipo M2 ejerce un efecto inotrópico negativo asociado a un incremento en la producción de ON. Este mecanismo parece ser secundario a la estimulación del ciclo de los fosfoinosítidos vía la activación de la fosfolipas C; que desencadeando reacciones en cascada llevan a la producción de ON, el cual contribuye al efecto inotrópico negativo ejercido por bajas concentraciones de carbacol

Purification and partial characterization of an l-amino acid oxidase from bushmaster snake (Surucucu pico de jaca) Lachesis muta venom

L-amino-acid oxidase(L-AO) form the venom of Lachesi muta muta was purified 72 times (38%) by gel filtration on Sephadex G-100, followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-300. The protein was shown to be homogeneous by polyacrylamide gel electroforesis, immunoelectrophoresis, immunodiffusion and isoelectric focusing. Its specific activity was 44.4 units/mg protein, using 7.5 mM L-leucine as substrate a nd O-dianisidine as electron donor,at pH 7.6 and 25§C. The increase in absorbance at 436 nm was recorded. The enzyme was characterized as a glycoprotein with an S20,w=6.72,MW=138,000 and pI=5.2. It presented maxima at 389 and 460 nm and contained 2 mol of FAD per mole protein

Radiometric studies on the oxidation of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria

Um sistema radiométrico foi utilizado para estudar os padröes de oxidaçäo dos (U-C)L-aminoácidos por micobactérias sensíveis e resistentes a drogas. Foram usadas duas cepas do M. tuberculosis sensíveis a todas as drogas, H Rv e Erdman. As micobactérias resistentes foram M. tuberculosis H Rv resistente a 5 ug/ml de hidrazida, M. bovis, M. avium, M. intracellulare, M. kansasii e M. chelonei. As micobactérias foram inoculadas em frascos estéreis contendo o meio líquido 7H9 e um dos (U-C)L-aminoácidos. Cada micobactéria apresentou um padräo de oxidaçäo de aminoácidos, mas estes padröes näo foram suficientemente diferentes para identificá-la. Aminoácidos complexos como a prolina, fenilalanina e tirosina näo tiveram utilidade na identificaçäo das micobactérias, pois praticamente todos os microorganismos foram incapazes de oxidá-los. Nenhuma combinaçäo de aminoácidos foi capaz de separar as micobacterias sensíveis das resistentes a drogas

Alanine dehydrogenase in mycobacteria--a preliminary report.

Various mycobacterial species namely M. phlei, M. vaccae, M. scrofulaceum, M. avium and M. tuberculosis have been investigated for the presence of enzyme alanine dehydrogenase which could be important for utilization of alanine by TCA cycle. It was found that alanine dehydrogenase was present in all species of mycobacteria tested irrespective of the fact whether they are rapid or slow growers. Electrophoretic mobilities of alanine dehydrogenase from different species of mycobacteria were not found to be significant for taxonomical differentiation of rapid and slow growers.