L-Amino add oxidase

B. Geueke and W. Hummel, A new bacterial L-amino add oxidase with a broad substrate spedfidty purification and charaderization, Enzyme Microb. Technol. 2002b, 31, 77-87. 

Thus, using L-amino add oxidase from P. myxcfaciens and various amine-borane complexes or D-amino acid oxidase from porcine kidney and sodium cyanoboro-hydride, the preparation of several natural and non-natural enantiopure D- and L-amino adds was achieved, respectively [51]. In a more recent report, several P- and y-substituted a-amino adds were deracemized using D-amino add oxidase from Trigonopsis variahilis and sodium cyanoborohydride or sodium borohydride [52] (Scheme 13.20). 

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. 

The best substrates for ophidian L-amino acid oxidases are aromatic or, most generally, hydrophobic amino acids, polar and basic amino acids being deami-nated at much lower rates Glu, Asp, and Pro are not oxidized by L-amino add oxidase. L-Amino add oxidase is also active on ring-substituted aromatic amino acids, as well as on seleno cysteinyl derivatives. The substrate specificity depends on the source of the enzyme (e.g. Ophiophagus hannah L-amino acid oxidase also oxidizes Lys and Om) and on the pH. The of the reaction of Crotalus adaman-... 

L-Amino add oxidases. Small amounts of ammonia are generated by various L-amino acid oxidases, found in liver and kidney, that require a flavin mononucleotide (FMN) coenzyme. FMN is regenerated from FMNH2 by reacting with 02 to form H202. 

Snake venoms a mixture of toxins produced in the venom glands (parotid gland, or salivary gland of the upper jaw) of venomous snakes (asps or hooded snakes, e.g. the cobra sea snakes vipers, e.g. puff adder, rattlesnake). They consist of highly toxic, antigenic polypeptides and proteins (which cause paralysis and death of the prey), and enzymes (which facilitate the spread of the toxins, and initiate digestion of undivided swallowed prey). The enzymes indude hya-luronidase (promotes spread of toxins), ATPase and acetylcholine esterase (paralysis), phospholipases (hemolysis), proteinases and L-amino add oxidases (tissue necrosis and blood clotting). 

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. 

L-Amino add oxidase activity has been detected in a variety of moulds, yeasts and bacteria, but the enzymes from these sources have not been well characterised. A partly purified enzyme from Proteus vulgaris is curious in that it does not apparently produce hydrogen peroxide in the presence of molecular oxygen (equations 22-26) and absence of catalase. 

The enzymes catalysing this reaction are the L-amino add oxidases and the D-amino add oxidases. The role of the latter in metabolism has not yet been elucidated, forthenaturally occurring amino acids are generallyof the L-series. 

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. 

The preparation of D-amino acids with the above three-enzyme system requires enzymes with opposite stereochemical selectivity and a suitable amino add as a donor. While D-amino add oxidase is an enzyme, the function of which in Nature is mainly related to the ehmination of D-amino adds, L-amino acid oxidases are usually found in aggressive animals (snakes). Bacterial L-amino acid oxidases often show a specific activity that is too low for preparative purposes [33]. Moreover, D-amino transferases are less common than the L-specific ones and require more expensive D-amino adds as amino donors. 

Recently, L-amino acid deaminase (EC 1.4.3.x) activities have been identified, particularly from the Proteus genus [59]. This enzyme, constituted by 370 residues, is an FAD-containing L-amino acid oxidase flavoprotein that uses molecular oxygen to convert L-amino acids into the corresponding a-keto adds and ammonia but does not produce hydrogen peroxide. L-amino acid deaminase prefers amino acids with aliphatic, aromatic, and sulfur-containing side chains (the best substrates are L-heu, L-Phe, L-Met, and L-Trp) and its kinetic efficiency is quite low because of the low Vnm value (<2 units/mg protein). 

Caligiuri, A., D Arrigo, P., Rosini, E Tessaro, D., Molla, G., Servi, S. and Polle-gioni, L. (2006) Enzymatic conversion of unnatural amino adds by yeast D-amino add oxidase. Advanced Synthesis Catalysis, 348, 2183-2190. 

Trost, E.M. and Fischer, L. (2002) Minimization of by-product formation during D-aminoadd oxidase catalyzed racemate resolution of D/L-amino adds. Journal of Molecular Catalysis B ... 

L-amino acid oxidase FMN Catabolism of L-amino adds to keto acids... 

Pilone M S, Pollegioni L (2002) D-amino add oxidase as an industrial biocatalyst. Biocatal Biotransform 20 145-159... 

L-Amino acid oxidase of rat kidney appears to catalyze the same type of reaction as n-amino acid oxidase. It oxidizes the monoamino, mono-carboxylic acids, but does not attack either dicarboxylic or polybasic compounds. Proline and W-methylamino adds are oxidized. A unique property of this enzyme is its ability to oxidize hydroxy acids. Only L-hydroxy acids are attacked. The ratio of hydroxy acid oxidation rate to amino acid rate is constant throughout purification. In general, hydroxy acid oxidation proceeds somewhat more rapidly than amino add oxidation, and the hydroxy acids corresponding to the basic amino adds... 

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. ... 

Rat Kidney -h-Amino Add Oxidase Dehydrogenase). Studies by Krebs clearly indicated that different systems existed in animal tissues for the oxidation of n- and L-amino acids. However, while considerable success has been achieved in the purification of mammahan n-amino acid... 

The optical isomers of alanine have been prepared by amination of n- and v-a-bromopropionic acids. The latter have been prepared by the action of PC1, and acetyl chlo-nde on n-serine methyl ester hydrochloride (904), by tiie action of nitroqrl bromide on n-alanine (289), and by the resolution of nir -bromopropionic add (147,6S4-656,507, 845). The preparation of n-alanine by the action of yeast on nn-alanine has been described by Ehrlich (234). L-Alanine has been prepared by the actimi of takadiastase (408, 584) and n-amino add orddase (79, 229) obtained from sheep or hog kidneys. The last author (Behrmn) isolated 22 g. of nearly pure ir-alanine from the reaction products of 72 g. of on-alanine. The velodty of the oxidation of i>-alanine by o-amino add oxidase has been determined by Stadie and Zapp (7S2). 

This classification indicates that the L- and D-amino add oxidases have broad substrate specificities, whereas the other enzymes have a narrower specificity. For the purpose of simplicity the above clasdfication will be used. The amino acid oxidase systems may be subclassified according to the nature of the hydrogen acceptor under two categories, aerobic and anaerobic. The former have been more commonly referred to as amino acid oxidases, whereas the latter are frequently referred to as dehydrogenases. 

The role of the coupled transaminase-glutamic dehydrogenase stems in amino acid nthesis has been discussed in Section IV, C. The possible role of L-amino acid oxidase in amino add syntheds has recently been proposed by Radhakrishnan and Meister ). However, the same objections raised above in relation to the low activity of this enzyme and its limited distribution must be answered before the role of this enzyme in amino acid biosyntheds can be conddered. 

Norvaline is strongly ketogenic (79). The L-form is attacked by L-amino acid oxidase (77) and the D-form by D-amino add oxidase (1S9). Its susceptibility to transamination has not been reported. Hassan and Greenberg (1S6) demonstrated that it is readily oxidized to COt in the intact animal. More of this amino add is excreted unchanged in the urine than is leudne. 

PoUegioni, L. and Molla, G. (2011) New biotech applications from evolved D-amino add oxidases. Trends Biotechnol., 29, 276-283. 

Another interesting pathway to defense metabolites is illustrated by escapin in the defensive secretion of the sea hare Aplysia califomica. Escapin is an L-amino acid oxidase which oxidizes L-lysine in an equilibrium mixture of antipredatory and antimicrobial metabolites which depends on the pH. This enzymatic oxidation led to A -piperidine-2-carboxylic acid and H2O2 as the major components of the mixture. Then, a nonenzymatic reaction occurs between H2O2 and the former metabolites resulting in two end products, 8-aminovaleric add and 8-valer-olactam (Kamio et al., 2009). 

A combination of D-amino acid oxidase and L-amino transferase is an example of a deracemization by stereoinversion. The product is an L-amino acid. The reaction catalyzed by amino transferase has an equilibrium constant close to unity, a very unpractical situation leading to uncomplete transformation and to the production of almost inseparable mixtures of amino acids (at least two, the amino acid product and the amino add used as an amino donor). For preparative purposes it is therefore mandatory to shift the equihbrium to the product side. A recent example of a deracemization procedure based on this coupled enzymatic system is the preparation of L-2-naphthyl-alanine 6 as illustrated in Scheme 13.9 [28]. The reaction occurs in one pot with initial oxidation of the D-amino acid catalyzed by D-amino acid oxidase from Rhodotonda gracilis. The hydrogen peroxide that is formed in stoichiometric amounts is decomposed by catalase. The a-keto add is the substrate for L-aspartate amino transferase (L-Asp amino transferase), which is able to use L-cysteine sulfinic acid 7 as an amino donor. 

In a similar way, D,L-pipecoUc add was converted to L-pipecolic acid [49]. However, the use of sodium borohydride, which slowly decomposes in aqueous solution, causes a significant rise in the pH value, which has to be controlled and which interferes with the necessary repeated cycles. Turner and Fotheringham expanded the applicability of the method, using milder and water stable reducing agents [50] and applying amino acid oxidases with either D- or L-spedficity. 

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