Aldoxime-converting enzymes

Aldoximes are prepared from aldehydes and hydroxylamine by condensation reaction, and the dehydration reaction of aldoxime is one of the most important methods of nitrile synthesis in organic chemistry. We speculated that it would become one of the most important examples in Green Chemistry if the dehydration reaction could be realized by an enzymatic method, and started studies on a new enzyme, aldoxime dehydratase, and its use in enzymatic nitrile synthesis. Furthermore, we clarified the relationship between aldoxime dehydratase and nitrile-degrading enzymes in the genome of the microorganisms and the physiological role of the enzyme. 

Isolation of microorganisms having aldoxime dehydratase activity  

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Notably, nitrile-degrading enzymes (e.g. nitrilase that converts the CN group to carboxylic acid, and nitrile hydratase that produces an amide function) have been described, and they co-exist with aldoxime-degrading enzymes in bacteria (Reference 111 and references cited therein). Smdies in this area led to the proposal that the aldoxime-nitrile pathway, which is implemented in synthesis of drugs and fine chemicals, occurs as a natural enzymic pathway. It is of interest that the enzyme responsible for bacterial conversion of Af-hydroxy-L-phenylalanine to phenacetylaldoxime, an oxidative decarboxylation reaction, lacks heme or flavin groups which are found in plant or human enzymes that catalyze the same reaction. Its dependency on pyridoxal phosphate raised the possibility that similar systems may also be present in plants . 

Table I presents data from McFarlane al, (8) showing that the multi-step oxidation of L-tyroslne to p-hydroxymandelonltrlle requires only 02 and a source of reducing power, most effectively supplied by an NADFH generating system. The table also shows that some of the L-tyroslne Is converted to p-hydroxyphenylacetaldoxime (aldoxime) as well as a trace of p-hydroxyphenylacetonltrlle (nitrile). The production of the aldoxime and nitrile In this experiment was the first Indication that the microsomal preparation from sorghum might constitute an organized enzyme entity Involved In the synthesis of dhurrln. If this were true, it should be possible to demonstrate each individual step of the reaction sequence between tyrosine and p-hydroxymandelontrlle.

Many of the enzyme systems of the biosynthetic process have now been studied. A microsomal fraction from etiolated sorghum seedlings converted L-tyrosine (2) into p-hy-droxymandelonitrile (3) (Halkier and Mpller, 1991 Mpller and Poulton, 1993). The particles required only NADPH in addition to the substrate.. /V-Hydroxytyrosine (4) was shown to be an intermediate in the synthesis of the aldoxime, and an N-hydroxylating cytochrome P-450 was identified (Dewick, 1984 Halkier and M0ller, 1989,1991 Halkier et al, 1991). The A -hydroxytyrosine (4) is converted to 2-nitro-3-(p-hy-droxyphenyl)propionic acid (5), which is, in turn, converted to l-ad-nitro-2-(p-hydroxyphenyl)ethane (6). The last compound appears to be the immediate precursor to ( )-p-hy-... 

Figure 2.4 Summary of glucosinolate biosynthesis. Amino acids or elongated amino acids are converted to aldoximes by the action of enzymes encoded by the CYP79 gene family. The aidoxime conjugates with cysteine, which acts as the sulphur donor, which is then lysed and detoxified by glycosylation and sulphation. The MAM and CYP79 enzymes have a high degree of specificity to the amino acid structure, whereas other enzymes of the pathway have lower specificity.

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