Extradiol cleavage

Nitrotoluene is degraded by a strain of Mycobacterium sp. via the corresponding 4-amino-3-hydroxytoluene (Spiess et al. 1998) this is dimerized abiotically to form a dihydrophenox-azinone, and after extradiol cleavage to 5-methylpyridine -2-carboxylate (Figure 2.2d). 

Block DW, F Lingens (1992b) Microbial metabolism of qninoline and related compounds XIV. Purification and properties of lH-3-hydroxy-4-oxoquinoline oxygenase, a new extradiol cleavage enzyme from Pseudomonas putida strain 33/1. Biol Chem Hoppe-Seyler 370 343-349. 

It is interesting to note that dioxygenases catalyzing the intradiol cleavage contain the ferric form of iron as the sole cofactor, whereas those catalyzing the extradiol cleavage contain the ferrous form. 

Table 3 compares some key properties of the enzyme-substrate complexes of the intradiol and extradiol cleaving dioxygenases. A mechanism for extradiol cleavage must account for the differing metal requirements of the two classes of enzymes and their distinct regiospecificities. A key difference is the reactivity of the ES complexes toward NO. Whereas the ES complexes of extradiol enzymes readily react with NO to form ES—NO adducts, those of intradiol enzymes do not react with NO unless the Fe(III) center is reduced prior to exposure to NO [167], Thus the Fe(II) center in the extradiol cleaving enzymes appears... 

Figure 18 Proposed mechanism for the extradiol cleavage of catechol.

A mechanism for extradiol cleavage is proposed in Figure 18 [5,158], Substrate binds first to the iron(II) center, followed by 02, to form a ternary complex akin to the ES—NO complex described earlier. Electron transfer from metal to 02 in the Fe(II)—02 adduct results in a superoxide-like moiety and imparts nucleophilic character to the bound 02. This in turn generates semiquinone character on the bound substrate, which is attacked by the nascent superoxide to form a peroxy intermediate that decomposes by a Criegee-type rearrangement to the observed product. 

The distal extradiol cleavage of L-dopa 12, catalyzed by an iron-dependent dioxygenase, gives an alanyl muconic semialdehyde derivative 18 which, on cyclization and lactol oxidation, yields stizolobic acid 16. The pyrone ring is then ammonolyzed24 to give 3-(6-carboxy-2-oxo-4-pyridyl)alanine 17 (Scheme 3). 

The relative rates of reaction for these substrates compared to catechol are as follows catechol, 100 3-methylcatechol, 8 3-methoxycatechol, 0.8 o-aminophenol, 0.1. We note that electron donating groups seem to be required for extradiol cleavage and that as the reaction becomes slower relative to catechol, the proportion of extradiol cleavage increases. 

Component 13 has been suggested to arise from prior hydroxylation of the ring followed by intradiol cleavage and lactonization. Though plausible, this mechanism does not satisfactorily explain why 15 is not observed at all in several cases. A more reasonable suggestion involves the extradiol cleavage of the ring, subsequent oxidation of the aldehyde function, and lactonization. 

Finally, it is interesting to note that only 13 is formed in cases 1 and 8 and only 15 is formed in cases 3,4, and 9. If 13 arises from extradiol cleavage while 15 results from intradiol cleavage, the above experiments have provided clues as to how such specific cleavages may be effected by the active sites of dioxygenases. 

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