Nitroalkane oxidase (NAO) catalyzes the oxidation of neutral nitroalkanes to aldehydes and ketones with oxygen consumption and the production of hydrogen peroxide and nitrite. The enzyme is a flavoprotein from the fungus Fusarium oxysporum. The active site base, Asp402, abstracts one proton from the substrate to give a carbanion which then attacks the flavin adenine dinucleotide (FAD). The three dimensional crystal structure of NAO shows that Arg409 is 3.6 Å from Asp402. When Arg409 is mutated to Lys, the rate constant for proton abstraction decreases 100-fold. The three-dimensional structure of NAO also reveals the existence of a tunnel which extends from the protein exterior and terminates at the FAD N5 atom and the residues Asp402 and Phe401. We mutated amino acids in the tunnel into tryptophan, phenylalanine and leucine. The L99W, S276W and S276A enzymes showed the biggest decreases in both kcat and kcat/Km; these amino acids are closest to the FAD molecule and the active site. Mutation of amino acids farther away from the active site showed very small changes in the kinetic parameters. Ser276 is hydrogen bonded to Asp402 in the wild-type enzyme. When this amino acid is mutated to alanine or tryptophan, k3, the rate constant for proton abstraction, decreases around 35 fold. Asp402, Arg409 and Ser276 constitute a catalytic triad in the active site of nitroalkane oxidase, and both Arg409 and Ser276 are important for positioning Asp402 and catalysis.

The flavoprotein spermine oxidase catalyzes the oxidation of spermine and oxygen to spermidine, 3-aminopropanol, and hydrogen peroxide. To allow mechanistic studies of the enzyme, methods have been developed to obtain large amounts of purified recombinant protein. The enzyme requires co-expression with chaperone proteins GroEL and GroES to remain soluble and active. Purification requires the use of a Ni-NTA and size exclusion column. Human spermine oxidase is a monomer with an extinction coefficient of 14000 M-1cm-1. The kinetic mechanism is ping pong. Therefore, oxygen is bound to the enzyme before spermidine is released. N1-Acetyl spermine is a slow substrate with kcat and kcat/Km values 2 and 3 orders of magnitude smaller than the values for spermine. Spermidine is a competitive inhibitor, and 1,8-diaminooctane (DAO) is an uncompetitive inhibitor. The pH effects indicate that two ionizable groups are present in the kcat/Km profile and one ionizable group is in the kcat profile. The reductive half reaction reveals no phase other than the reduction of the FAD, indicating the probability of a single chemical step. Reduction is not limiting to the overall reaction. Isotope effects were determined; Dkcat at pH 7.5 = 4.1±0.4, pH 8.5 = 2.6±0.01.

Ethylene, a plant hormone, plays an essential role in many aspects about plant development, growth, ripening, and senescence. In addition, it also regulates several responses when plants suffer stress from drought, flood, herbivore bites, wound, etc. ACC synthase and ACC oxidase belong to two multigene families. In this study, PCR (polymerase chain reaction) and RACE (rapid amplification of cDNA ends) methods were used to amplify the ACC oxidase sequences in Cattleya bicolor orchid flower. The results show that there exists differences in the 3¡¦-UTR (untranslated region) of orchid gene sequences. Compare the ACC oxidase sequences, including the cDNA ORF (open reading frame) sequences and the amino acid sequences, of several different species, the sequence similarity among the three Laeliinae orchids, namely C.bicolor, C. intermedia, and Laelia anceps, is the highest. The similarity of cDNA ORF sequences and amino acid sequences between orchids and the other plants, such as rice, apple and torenia, is comparatively lower. It was proposed that the protein located in cytoplasma (or in mitochondrial matrix space), agrees with the result from analysis of amino acid hydrophilicity prediction. The ultimate goal of this study is to postpone the flower senescence by the way of plant transfection. In the present findings, it only deals with the cloning and analysis of the ACC oxidase genes in C. bicolor.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 125-132). Available also in a digital version from Dissertation Abstracts.

No description available.

Alcoholism is a prevalent multifactorial disease with both genetic and environmental components. Monoamine oxidase (MAO) has been proposed as a susceptibility marker for familial alcoholism but consistent evidence of either specific MAO variants in alcoholics or allelic segregation in at-risk families has not been presented. Two structural genes on the X chromosome encode two forms of the enzyme, MAO-A and MAO-B. Kinetic constants for platelet MAO-B and restriction fragment length polymorphisms for MAO-A were determined in alcoholics with multigenerational family histories of alcoholism, high risk relatives of familial alcoholics and low risk controls with no family history of alcoholism. Mean elevated levels of MAO-B deamination were observed in alcoholics and high risk individuals. Alcoholic and high risk individuals did not differ from non-alcoholics with respect to MAO-B tryptamine affinity or MAO-A polymorphisms. Significant non-genetic factors influence MAO-B activity. MAO variants are unlikely to define a genetic predisposition to alcoholism.

Phenol oxidases mediate the degradation of recalcitrant compounds, polyphenolics, in wetland soils and are considered to play a key role in the microbial carbon cycle of peatlands which predominate in boreal biomes. In order to validate a method for quantification of oxidative enzyme activity in acidic wetland environments, the relationship between pH and substrate oxidation was studied using the standard enzyme tyrosinase and in soils collected from six freshwater wetlands including three marshes in north Florida and peatlands of northern Minnesota. Phenol oxidase (PO) activity was quantified with two commonly used assay substrates, ABTS (2,2'-azino-bis(3-ethylobenzthiazoline-6-sulfonic acid) and L-DOPA (L-3,4-dihydroxyphenylalanine), across a pH range of 4 to 7 which matched the in situ pH range of the studied wetlands. The PO assay is sensitive and activity could be detected with either substrate across a pH range of 4 to 7. However, with the standard enzyme tyrosinase, it was shown that a large change or threshold in oxidation rates occurred at pH 5. At pH < 5, L-DOPA oxidation rates were greatly diminished and ABTS oxidation was at a maximum. Above pH 5, ABTS oxidation occurred at much slower rates and L-DOPA oxidation was at a maximum. The pH response of PO activity in wetland soils corroborated observations made with tyrosinase. Thus, ABTS is recommended to be an effective substrate for the quantification of PO activity at an in situ pH of < 5, while L-DOPA is recommended at an in situ pH of > 5. In soils collected from a northern Minnesota peatland, assays conducted at an in situ pH of 4 showed one to two orders of magnitude higher rates of PO activity in solid phase peat in comparison to porewater, indicating that the majority of PO activity is associated with the peat. At three Minnesota peatland sites, PO activity was shown to attenuate with depth in agreement with the activities of other enzymes and with rates of peat decomposition.

No description available.

No description available.

Polyphenol oxidases are important enzymes because of their role in food spoilage, oxidizing monophenols into o-diphenols and/or diphenols into the corresponding o-quinones. The resulting compounds are unstable and can rapidly form brown colored compounds, called melanins. Polyphenol oxidases, (PPOs) have been purified from several sources, particularly from fruits and vegetables. However, successful purification of PPO to homogeneity from plant sources has always been difficult. / The purification procedure of PPOs from cassava tuber consisted of (1) the preparation of cassava acetone powder; (2) the buffer extraction of the acetone powder to obtain a crude extract, followed by one of two possible purification procedures. The first consisted of ammonium sulfate fractionation, ion exchange chromatography on Mono-Q and gel filtration on Superdex G75 to yield two isoenzymes, PPO1 and PP02 having molecular weights of 71.8 +/- 6.0 and 69.6 +/- 1.5 kDa, respectively. The second purification procedure involved hydrophobic interaction chromatography (HIC) on phenyl-sepharose CL-4B followed by gel filtration on Superdex G75 to yield a single active PPO fraction of 68.3 +/- 2.8 kDa molecular weight. / The two isoenzymes obtained by ion exchange chromatography exhibited pH optima of 6.5 (PPO1) and 6.8 (PPO2) and were stable in the pH range of 7.5 to 10.0. These two isoenzymes had a temperature optimum of 30--40°C. PPO2 retained 65% of its original activity after heating at 50°C for 10 min whereas PPO1 was completely inactivated by the same treatment. The PPO fraction obtained by HIC purification exhibited a pH optimum of 7.5 with catechol and D,L-dopa as substrates and was stable in the pH range 4 to 8. Its temperature optima, were 20 and 30°C respectively with D,L-dopa and catechol as substrates and this PPO fraction was able to retain 80% of its original activity after heating at 50°C for 10 min. Unstable enzymes were obtained by the ion exchange chromatography purification procedure suggesting that artifacts were created. / Kinetic studies performed with the PPO fraction obtained by the HIC purification showed that catechol had the highest catalytic efficiency ratio. The Km values were 28.1, 5.27 and 3.72 mM for catechol, catechin and D,L-dopa, respectively. The PPO from the HIC purification procedure was inhibited by benzoic acid and p-coumaric acid and inactivated by diethyldithiocarbamate but not by EDTA. L-Cysteine, ascorbic acid and its derivatives (erythorbic acid and sodium erythorbate) were also inhibitors of the oxidation of catechol, catechin and D,L-dopa.