Bacteria amino acid oxidases

One of the most frequently neglected rules in the investigation of modes of action pertains to the use of excessive drug concentrations. In a study of the effects of chlortetracycline on a (mammalian) D-amino acid oxidase, 6 the antibiotic concentration was 1.2 x 10 3M which is 575 jig/ml, Le. more than two orders of magnitude higher than the growth-inhibitory concentrations for susceptible bacteria. 

The enzymes discussed in this paragraph are D- and L-amino acid oxidases, the presence of which has been detected in numerous organisms and tissues. They were only recently identified in bacteria and the corresponding enzymes were isolated (for a review see [54]). These enzymes are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of amino acids (Scheme 13.21). 

L-Amino acid oxidase is a flavoenzyme that catalyzes the oxidative deamination of L-amino adds. L-Amino acid oxidase activities have been detected in mammals, birds, reptiles, invertebrates, molds, and bacteria [54]. L-Amino acid oxidases show the typical absorption spectrum due to the presence of a molecule of non-covalently bound FAD per subunit (with maxima at 465 and 380nm) they behave like flavoprotein oxidases, as in the case of D-amino acid oxidase. L-Amino add oxidase isolated from rat liver was reported to utilize flavin mononudeotide (FMN) as a co-enzyme, but since it is more active on L-hydroxy acids than on amino adds, it was thus considered as an L-hydroxy add oxidase. Even a partially purified L-amino acid oxidase from turkey Uver appeared to have FMN as a co-factor. 

D-Amino acid oxidase occurs in peroxisomes containing other enzymes that produce H2O2 (e.g., L-a-hydroxy acid oxidase, citrate dehydrogenase, and L-amino acid oxidase) and catalase and peroxidase, which destroy H2O2. In leukocytes, killing of bacteria involves hydrolases of lysosomes and production of H2O2 by NADPH oxidase (Chapter 15). Conversion of D-amino acids to the corresponding a-keto acids removes the asymmetry at the a-carbon atom. The keto acids may be aminated to L-amino acids. By this conversion from D- to L-amino acids, the body utilizes D-amino acids derived from the diet ... 

In a first step the chiral center at the a-aminoadipyl side-chain has to be eliminated. Besides commercially available D-amino acid oxidase (DAO) from pig kidney for laboratory use, the enzyme is also synthesized by bacteria, yeasts and fungi. This enzyme can be used for the resolution of D,L-amino acids in various solutions, but of particular interest is its capacity for oxidative deamination of Ceph C. The first research was carried out by Glaxo Laboratories Ltd. [11] where DAO isolated from different fungal cells was used. Under the reaction conditions used, in the presence of oxygen as a co-substrate, the enzyme deaminates cephalosporin C to give a-ketoadipyl-7-ACA, ammonia and hydrogen peroxide (Fig. 5). 

Microorganisms including yeasts and bacteria are able to produce D-amino acid oxidases, and interest has centered particularly on the use of /7-alkane as growth substrates (Kawamoto et al. 1977). The enzyme has achieved importance for its role in carrying out the first step in the transformation of cephalosporin C to 7-aminocephalosporanic acid that is an intermediate for the synthesis of semisynthetic cephalosporins. The nucleotide sequence of the enzyme from Rhodotorula gracilis ATCC 26217 has been established and the gene could be overexpressed in Escherichia coli (Alonso et al. 1998). 

D-amino acid oxidase will oxidize only serine having R configuration at C(2). Glycolate oxidase will remove only the pro-R hydrogen of glycolic acid. Does the product (0=CHC02H) contain tritium Explain your reasoning, b. Enzymatic oxidation of naphthalene by bacteria proceeds by way of the intermediate aT-diol shown. Which prochiral face of C(l) and C(2) of naphthalene is hydroxylated in this process ... 

Amino acid oxidase, Aspergillus oryzae enzyme, Chymotrypsin, Diastase, Emulsin, Hexokinase, Gluco-syltransf erase, Hog kidney enzyme, Iso-merase. Papain, Phosphatase, Pseudomonas extract Microorganisms Soil bacteria Acetobacter sub-oxydans. Streptococcus faecalis Fungi (Mold), Yeast CN-... 

L-Amino Add Oxidase. In 1944 L-amino acid oxidases were described in 3 sources animal kidney, snake venom, and bacteria. The enzyme was also detected in animal liver. The purification from rat kidney required large amounts of tissue because of the low activity. A 200-fold purification gave a preparation believed to be essentially pure. This protein has a molecular weight somewhat over 120,000, and contains 2 flavin mononucleotides per mole. If the protein isolated is indeed the enzyme, it has one of the lowest turnover numbers determined only 6 molecules of amino acid are oxidized per molecule of protein per minute, whereas n-amino add oxidase has a turnover number of more than 1000. 

The ability to oxidize L-amino adds is widespread among bacteria, but the enzymes responsible have not been well characterized. Proteus vulgaris is able to oxidize a large series of L-amino acids, but on storage at 0 C. loses the ability to oxidize 9 out of 20 amino acids tested. The relative rates of oxidation of the other 11 amino adds do not remain constant. An active cell-free preparation was obtained, but it is not known how many amino acid oxidases may exist in these organisms. ... 

As we have just seen, L-glutamic acid is not deaminated by the action of the L-amino acid oxidase of animal tissues and bacteria. But, in the presence of a specific enzyme, glutamic dehydrogenase, it undergoes oxidative deamination in the presence of either DPN or TPN. This reversible reaction gives a-iminoglutaric acid. 

Both optical isomers of a DL-amino acid are oxidised to a-keto acids and other products by the action of molds, yeasts, bacteria and amino acid oxidases but one form of some of the amino adds is oxidised more rapidly than the other. Either the d- or n-amino acid may be isolated from the reaction products depending upon the t3rpe of organism or amino acid oxidase employed. Procedures for the preparation of various d- and L-amino acids in this manner are outlined in the footnotes to Table I. The literature on this topic prior to 1922 has been reviewed by Ehrlich (239). 

A. L-Amino Acid Oxidase. Snake venom amino acid oxidase is different from microorganism (bacteria and fungi) enzymes in that the former acts of levorotary-amino acids, and the latter work on dextrorotary-amino acids. The enzyme converts free amino acids into a-keto acids. 

The natural amino acids are mainly a-amino acids, in contrast to (3-amino acids such as p-alanine and taurine. Most a-amino acids have four different substituents at C-2 (Ca). The a atom therefore represents a chiral center—I e., there are two different enantiomers (L- and D-amino acids see p. 8). Among the proteinogenic amino acids, only glycine is not chiral (R = H). In nature, it is almost exclusively L-amino acids that are found. D-Amino acids occur in bacteria—e. g., in murein (see p.40)—and in peptide antibiotics. In animal metabolism, D-Amino acids would disturb the enzymatic reactions of L-amino acids and they are therefore broken down in the liver by the enzyme D-amino add oxidase. 

Limited amino acid sequence information has shown that long-chain a-hydroxyacid oxidase from rat kidney is also related to these FMN-containing oxidoreductases (55). It is likely that several further members of this family remain to be identified. The flavodehydrogenase domain shows no sequence similarity to the lactate dehydrogenase from bacteria and higher eukaryotes that utilize NAD as a substrate. Yeasts lack such an enzyme and the substrate specificity of flavocytochrome 62 has presumably evolved independently of the NAD-linked dehydrogenases. 

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