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.

No abstract

1. A bacterium, Achromobacter D, isolated from garden soil by elective culture, utilized N-methylisonicotinic acid (4-carboxy-1-methylpyridinium chloride) as sole carbon source. 2. Extracts of N-methylisonicotinate-grown cells oxidized this substrate only after supplementation with a source of nicotinamide nucleotides and then consumed 1 mol of O(2) and released 1 mol of CO(2)/mol of N-methylisonicotinate supplied. 3. The N-methyl group of the substrate was released as methylamine whereas the five C atoms of the pyridine ring were accounted for as succinate and formate. The CO(2) evolved by extracts was believed to derive from the carboxyl group on C-4 of the heterocyclic ring. 4. The immediate precursor of the succinate end-product was succinic semialdehyde; the inducible nature of succinic semialdehyde dehydrogenase in N-methylisonicotinate-grown cells supported this finding. 5. There was no evidence for monohydroxylation of the ring, but the time sequence of the appearance of the end-products indicated that the oxygen-requiring, NADH-requiring and decarboxylation steps clearly preceded the formation of methylamine and succinate. 6. The results are consistent with the oxidative cleavage of a partially reduced heterocyclic ring followed by several hydrolytic and dehydrogenase steps resulting in the appearance of the end-products.

No abstract

The distance between aspartokinase and homoserine dehydrogenase active sites was determined using fluorescence energy transfer between modified substrates. The fluorescent 1,N(6)-ethenoadenosine 5'-triphosphate was bound at the kinase active site by Co(III) affinity labeling. Reduced thionicotinamide adenine dinucleotide phosphate quenched the fluorescence of bound nucleotide. Fluorescence depolarization measurements led to a delimitation of the value of the dipolar orientation factor to the range 0.3 to 2.8. The distance between the fluorescent probe and the quencher was 29 +/- 4 A. In the presence of threonine, this distance increased to 36 +/- 5 A. Threonine binding either increased the intersite distance by ca. 7 A or caused a reorientation of the probe at the dehydrogenase site.