1. Several species of micro-organisms that were capable of utilizing pyridine compounds as carbon and energy source were isolated from soil and sewage. Compounds degraded included pyridine and the three isomeric hydroxypyridines. 2. Suitable modifications of the cultural conditions led to the accumulation of pyridinediols (dihydroxypyridines), which were isolated and characterized. 3. Three species of Achromobacter produced pyridine-2,5-diol from 2- or 3-hydroxypyridine whereas an uncommon Agrobacterium sp. (N.C.I.B. 10413) produced pyridine-3,4-diol from 4-hydroxypyridine. 4. On the basis of chemical isolation, induction of the necessary enzymes in washed suspensions and the substrate specificity exhibited by the isolated bacteria, the initial transformations proposed are: 2-hydroxypyridine --> pyridine-2,5-diol; 3-hydroxypyridine --> pyridine-2,5-diol and 4-hydroxypyridine --> pyridine-3,4-diol. 5. A selected pyridine-utilizer, Nocardia Z1, did not produce any detectable hydroxy derivative from pyridine, but carried out a slow oxidation of 3-hydroxypyridine to pyridine-2,3-diol and pyridine-3,4-diol. These diols were not further metabolized. 6. Addition of the isomeric hydroxypyridines to a model hydroxylating system resulted in the formation of those diols predicted by theory.

1. Pyridine-3,4-diol (3,4-dihydroxypyridine, 3-hydroxypyrid-4-one), an intermediate in 4-hydroxypyridine metabolism by an Agrobacterium sp (N.C.I.B. 10413), was converted by extracts into 1mol of pyruvate, 2mol of formate and 1mol of NH(3) at pH7.0. 2. Formate, but not the alternative likely product formamide, was further oxidized fivefold faster by 4-hydroxypyridine-grown washed cells than by similar organisms grown on succinate. 3. The oxidation of pyridine-3,4-diol by crude extracts at pH8.5 required 1mol of O(2)/mol of substrate, produced 1mol of acid and led to the formation of formate and a new compound with an extinction maximum of 285nm (Compound I). This step was believed to be mediated by a new labile dioxygenase (t((1/2))=4h at pH7.0, 4 degrees C) cleaving the pyridine ring between C-2 and C-3. 4. Many of the properties of this pyridine-3,4-diol dioxygenase paralleled those of the extradiol (;meta') oxygenases of aromatic-ring cleavage. The extreme lability of the enzyme has so far precluded extensive purification. 5. Compound I showed changes in the u.v.-absorption spectrum with pH but after acidification it was converted into a new product, 3-formylpyruvate, with an extinction maximum now at 279nm. 6. Both Compound I and 3-formylpyruvate were metabolized by extracts but at very different rates. The slower rate of metabolism of Compound I was nevertheless consistent with that of pyridine-3,4-diol metabolism. 7. On acidification Compound I released about 0.65mol of NH(3) and has been identified as 3-formiminopyruvate. 8. 3-Formylpyruvate was hydrolysed to formate and pyruvate (K(m) 2mum) by an acylpyruvate hydrolase active against several other dioxo homologues. The activity of this enzyme was much lower in extracts of succinate-grown cells.

1. Washed suspensions of two Achromobacter species (G2 and 2L), capable of growth upon 2- and 3-hydroxypyridine respectively as sources of C and N, rapidly oxidized their growth substrate pyridine-2,5-diol (2,5-dihydroxypyridine) and the putative ring-cleavage product maleamate without a lag. Suspensions derived from fumarate plus (NH(4))(2)SO(4) cultures were unable to do so. 2. Extracts of both bacteria oxidized pyridine-2,5-diol with the stoicheiometry of an oxygenase forming 1mol of NH(3)/mol of substrate. 3. Heat-treated extracts, however, formed maleamate and formate with little free NH(3). 4. The conversion of maleamate into maleate plus NH(3) by extracts of strain 2L, fractionated with (NH(4))(2)SO(4), and the metabolism of maleamate and maleate to fumarate by extracts of both strains demonstrated the existence of the enzymes catalysing each reaction of the maleamate pathway in these bacteria. 5. The pyridine-2,5-diol dioxygenase (mol.wt. approx. 340000) in extracts of these Achromobacter species required Fe(2+) (1.7mum) to restore full activity after dialysis or treatment with chelating agents; the enzyme from strain 2L also had a specific requirement for l-cysteine (6.7mm), which could not be replaced by GSH or dithiothreitol. 6. The oxygenase was strongly inhibited in a competitive manner by the isomeric pyridine-2,3- and -3,4-diols.

1. The first metabolic step in the biodegradation of 4-hydroxypyridine by an Agrobacterium sp. was hydroxylation to form pyridine-3,4-diol. 2. Extracts required 1mol of O(2) and 1mol of NADH or NADPH for the conversion of 4-hydroxypyridine into pyridine-3,4-diol, suggesting that the enzyme responsible, 4-hydroxypyridine-3-hydroxylase, was a mixed function mono-oxygenase. 3. After treatment with acidic (NH(4))(2)SO(4) the enzyme required FAD for activity; FMN and riboflavin would not substitute for FAD. 4. The rate of anaerobic reduction of FAD by NAD(P)H was increased more than tenfold in the presence of 4-hydroxypyridine, suggesting that the mechanism of hydroxylation was similar to that of other aromatic hydroxylases which are of the mono-oxygenase type. 5. The partially purified enzyme was extremely specific for its heterocyclic substrate but would utilize either NADH or NADPH. 6. 4-Hydroxypyridine-3-hydroxylase was strongly inhibited by high substrate concentration (above 0.5mm) especially below pH7.5. 8. The inflexion at pH8.4 in a pK(m) versus pH plot, together with strong inhibition by p-chloromercuribenzoate, suggested a role for thiol groups in substrate binding.

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