~ ~Four Gram-positive bacteria have been isolated from separate soil samples by enrichment culture with acetone as sole source of carbon. Whole cells of all strains grown on acetone rapidly oxidized acetone, acetol and methylglyoxal, and three of the four also oxidized isopropanol. The patterns of induced enzymes in cell extracts are compatible with the oxidation sequence : isopropanol -+ acetone -+ acetol -+ methylglyoxal -+ pyruvate. Although an enzyme system capable of converting acetone into acetol has not been detected, the inclusion of acetol in the pathway is supported by the results of studies with whole cells and [14C]acetone. The proposed pathway of acetone metabolism is contrasted with evidence for an alternative, but not fully understood, pathway used by Mycobacterium vaccae JOB5. I N T R O D U C T I O NAlthough several groups have studied acetone degradation by animals and microorganisms, the precise metabolic routes involved and the nature of the carbon fragments that enter the central metabolic pathways are still unclear. Sakami & Lafaye (1951) and Rudney (1954) reported studies with the rat which indicated a cleavage of acetone metabolites to yield acetate and formate while pyruvate and lactate were formed directly. Studies with micro-organisms have also indicated the probable cleavage of the acetone carbon skeleton into organic C, and Cz fragments. Early work (Supniewski, 1923 ;Goepfert, 1941) indicated the formation of formate and formaldehyde by species of Bacillus and Fusarium. The oxidation of acetone by a soil diphtheroid (Levine & Krampitz, 1952) yielded acetaldehyde and a C, fragment. Vestal & Perry (1969) showed that acetone was an intermediate in propane metabolism by Mycobacterium vaccae JOB5 and that further metabolism yielded acetate and C02, resulting in the induction of isocitrate lyase (EC 4.1.3.1) in propane-grown cells. Lukins & Foster (1963) have, like others, provided evidence that acetol (1-hydroxyacetone) is an intermediate in acetone degradation. However, in their studies with Mycobacterium smegmatis, they presented no clear evidence to indicate how acetol is further metabolized.Previous workers have relied heavily on whole-cell oxidation studies and radiochemical techniques. In this study, extracts have also been used to investigate the enzymology of a number of isolates grown with acetone and related compounds as sole sources of carbon. A route for the conversion of acetone into a central metabolic pathway intermediate is proposed. M E T H O D SBacterial strains. Strains A1 and A2 were obtained from Aberystwyth soil and estuarine mud (Penarth), respectively, by elective culture with acetone (0.2 %, viv) as sole source of carbon. Strains SAl and SPl were obtained by elective culture with acetone and isopropanol, respectively, from soil samples taken in the
Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD(+) pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.
1. An organism that grows on nitrilotriacetate as sole source of carbon and energy was isolated in pure culture and was identified as a pseudomonad. 2. Cell-free extracts of the nitrilotriacetate-grown pseudomonad contain an enzyme that catalyses the NADH-and O(2)-dependent oxidation of nitrilotriacetate to iminodiacetate and glyoxalate. This enzyme is absent from extracts of glucose-grown cells. 3. Compared with growth on glucose, growth on nitrilotriacetate results in increased activities of enzymes of glycine and serine metabolism, namely serine hydroxymethyltransferase, glycine decarboxylase, serine-oxaloacetate aminotransferase and hydroxypyruvate reductase. 4. Cell-free extracts of the nitrilotriacetate-grown organism contain the enzyme glyoxalate carboligase and, when supplemented with NADH, Mg(2+) and thiamin pyrophosphate, can catalyse the anaerobic conversion of glyoxalate into glycerate. 5. These results are incorporated in a scheme which shows the oxidative metabolism of nitrilotriacetate by the successive removal of C(2) units to form 2mol of glyoxalate and 1mol of glycine per mol of nitrilotriacetate degraded. The glyoxalate and glycine are then both metabolized to glycerate by separate pathways, via tartronic semialdehyde and serine respectively. The role of this scheme in the growth of the organism on nitrilotriacetate is discussed.
The Arthrobacter sp. is able to grow with (+I-, (-)-1-phenylethanol or the racemic mixture as sole source of carbon. Growth is most rapid with the (-)-isomer, doubling time 12 h. Enzymes of the already established pathway for acetophenone oxidation are also induced by growth with 1-phenylethanol and in addition an induced 1-phenylethanol dehydrogenase that requires NAD as electron acceptor is formed.The 1-phenylethanol dehydrogenase is active with both 1-phenylethanol isomers but shows a marked preference for the (-)-isomer. Relative activity towards the two isomers is mirrored in the rates of growth with the two isomers and the oxidative capabilities of induced cells.,The metabolism of the isomers of 1 -phenylethanol by the Arthrohacter sp. is integrated into the established pathway for acetophenone oxidation.The pathway of acetophenone metabolism by Nocardia T5 shows no significant differences from that already established for metabolism of the compound by the Arthvobacter sp. The same spectrum of induced enzymes is formed and the evidence suggests that catechol oxidation again proceeds by 'ortho' fission and the P-oxoadipate pathway, the formation of even transient amounts of cis,cis-hydroxy muconic semialdehyde has never been observed.In contrast the metabolism of the isomers of 1-phenylethanol by Nocardia T5 is achieved by a quite different pathway. The side chain is retained without modification during hydroxylation to yield 3-(I '-hydroxyethy1)catechol. Ring cleavage, by 'meta fission' yields 2,7-dihydroxy-6-oxoocta-2,4-dienoate the further fragmentation of which yields lactate and equimolar amounts of pyruvate and acetaldehyde.Growth rates are identical, irrespective of which isomer of 1-phenylethanol is used as carbon source and the enzymes that convert 1-phenylethanol to vinyl pyruvate and lactate appear to display no specificity for that chiral centre.
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