The catabolism of phenylalanine to 2-phenylethanol and of tryptophan to tryptophol were studied by 13 C NMR spectroscopy and gas chromatography-mass spectrometry. Phenylalanine and tryptophan are first deaminated (to 3-phenylpyruvate and 3-indolepyruvate, respectively) and then decarboxylated. This decarboxylation can be effected by any of Pdc1p, Pdc5p, Pdc6p, or Ydr380wp; Ydl080cp has no role in the catabolism of either amino acid. We also report that in leucine catabolism Ydr380wp is the minor decarboxylase. Hence, all amino acid catabolic pathways studied to date use a subtly different spectrum of decarboxylases from the five-membered family that comprises Pdc1p, Pdc5p, Pdc6p, Ydl080cp, and Ydr380wp. Using strains containing all possible combinations of mutations affecting the seven AAD genes (putative aryl alcohol dehydrogenases), five ADH genes, and SFA1, showed that the final step of amino acid catabolism (conversion of an aldehyde to a long chain or complex alcohol) can be accomplished by any one of the ethanol dehydrogenases (Adh1p, Adh2p, Adh3p, Adh4p, Adh5p) or by Sfa1p (formaldehyde dehydrogenase.)

The metabolism of leucine to isoamyl alcohol in yeast was examined by 13 C nuclear magnetic resonance spectroscopy. The product of leucine transamination, ␣-ketoisocaproate had four potential routes to isoamyl alcohol. The first, via branched-chain ␣-keto acid dehydrogenase to isovaleryl-CoA with subsequent conversion to isovalerate by acyl-CoA hydrolase operates in wild-type cells where isovalerate appears to be an end product. This pathway is not required for the synthesis of isoamyl alcohol because abolition of branched-chain ␣-keto acid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isoamyl alcohol. A second possible route was via pyruvate decarboxylase; however, elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant did not decrease the levels of isoamyl alcohol produced. A third route utilizes ␣-ketoisocaproate reductase (a novel activity in Saccharomyces cerevisiae) but with no role in the formation of isoamyl alcohol from ␣-hydroxyisocaproate because cell homogenates could not convert ␣-hydroxyisocaproate to isoamyl alcohol. The final possibility was that a pyruvate decarboxylase-like enzyme encoded by YDL080c appears to be the major route of decarboxylation of ␣-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that at least one other unidentified decarboxylase can substitute to a minor extent.In most eukaryotes, the catabolism of the branched-chain amino acids leucine, isoleucine, and valine has been well understood for many years (1). The first step is a transamination in which ␣-ketoglutarate accepts the amino group (from leucine, isoleucine, and valine) producing glutamate and ␣-ketoisocaproic acid, ␣-keto-␤-methylvaleric acid and ␣-ketoisovaleric acid, respectively. Next is oxidative decarboxylation of the keto acids by branched-chain ␣-keto acid dehydrogenase to the corresponding acyl-CoA derivatives. Further steps yield, ultimately, acetyl-CoA and acetoacetate (from leucine), acetyl-CoA and propionyl-CoA (from isoleucine), and succinyl-CoA (from valine). All of these metabolites can enter the tricarboxylic acid (TCA) 1 cycle. It has been known for many years that yeasts do not operate the same metabolic routes because branched-chain amino acids can serve as the sole source of nitrogen but not carbon (2, 3). The predominant view, in a rather sparse literature, is that yeasts first use transamination but that decarboxylation of the keto acids proceeds via a "carboxylase" to an aldehyde that is then reduced in an NADH-linked reaction producing the appropriate "fusel" alcohol (2-4). This scheme is sometimes called the "Ehrlich pathway" to honor the originator of the ideas (5), which were slightly modified later (6). Acceptance of the so-called Ehrlich pathway is problematical for at least four reasons. First, the supposed pathway has never been proven to exist. Simply showing that e.g. radioactively labeled leucine is converted into isoamyl alcohol does not prove that the individual steps are those envisaged in ...

The metabolism of valine to isobutyl alcohol in yeast was examined by 13 C nuclear magnetic resonance spectroscopy and combined gas chromatography-mass spectrometry. The product of valine transamination, ␣-ketoisovalerate, had four potential routes to isobutyl alcohol. The first, via branched-chain ␣-ketoacid dehydrogenase to isobutyryl-CoA is not required for the synthesis of isobutyl alcohol because abolition of branchedchain ␣-ketoacid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isobutyl alcohol. The second route, via pyruvate decarboxylase, is the one that is used because elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant virtually abolished isobutyl alcohol production. A third potential route involved ␣-ketoisovalerate reductase, but this had no role in the formation of isobutyl alcohol from ␣-hydroxyisovalerate because cell homogenates could not convert ␣-hydroxyisovalerate to isobutyl alcohol. The final possibility, use of the pyruvate decarboxylase-like enzyme encoded by YDL080c, seemed to be irrelevant, because a strain with a disruption in this gene produced wild-type levels of isobutyl alcohol. Thus there are major differences in the catabolism of leucine and valine to their respective "fusel" alcohols. Whereas in the catabolism of leucine to isoamyl alcohol the major route is via the decarboxylase encoded by YDL080c, any single isozyme of pyruvate decarboxylase is sufficient for the formation of isobutyl alcohol from valine. Finally, analysis of the 13 C-labeled products revealed that the pathways of valine catabolism and leucine biosynthesis share a common pool of ␣-ketoisovalerate.

The metabolism of isoleucine to active amyl alcohol (2-methylbutanol) in yeast was examined by the use of 13 C nuclear magnetic resonance spectroscopy, combined gas chromatography-mass spectrometry, and a variety of mutants. From the identified metabolites a number of routes between isoleucine and active amyl alcohol seemed possible. All involved the initial decarboxylation of isoleucine to ␣-keto-␤-methylvalerate. The first, via branched chain ␣-ketoacid dehydrogenase to ␣-methylbutyryl-CoA, was eliminated because abolition of branched-chain ␣-ketoacid dehydrogenase in an lpd1 disruption mutant did not prevent the formation of active amyl alcohol. However, the lpd1 mutant still produced large amounts of ␣-methylbutyrate which initially seemed contradictory because it had been assumed that ␣-methylbutyrate was derived from ␣-methylbutyryl-CoA via acyl-CoA hydrolase. Subsequently it was observed that ␣-methylbutyrate arises from the non-enzymic oxidation of ␣-methylbutyraldehyde (the immediate decarboxylation product of ␣-keto-␤-methylvalerate). Mutant studies showed that one of the decarboxylases encoded by PDC1, PDC5, PDC6, YDL080c, or YDR380w must be present to allow yeast to utilize ␣-keto-␤-methylvalerate. Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol. This is the first demonstration of a role for the gene product of YDR380w, and it also shows that the decarboxylation steps for each ␣-keto acid in the catabolic pathways of leucine, valine, and isoleucine are accomplished in subtly different ways. In leucine catabolism, the enzyme encoded by YDL080c is solely responsible for the decarboxylation of ␣-ketoisocaproate, whereas in valine catabolism any one of the isozymes of pyruvate decarboxylase will decarboxylate ␣-ketoisovalerate.

Sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), formed by the hydrolysis of the plant sulfolipid, is a major component of the biological sulfur cycle. However, pathways for its catabolism are poorly delineated. We examined the hypothesis that mineralization of sulfoquinovose to inorganic sulfate is initiated by reactions of the glycolytic and/or Entner-Doudoroff pathways in bacteria. Metabolites of [U-13 C]sulfoquinovose were identified by 13 C-nuclear magnetic resonance (NMR) in strains of Klebsiella and Agrobacterium previously isolated for their ability to utilize sulfoquinovose as a sole source of carbon and energy for growth, and cell extracts were analyzed for enzymes diagnostic for the respective pathways. Klebsiella sp. strain ABR11 grew rapidly on sulfoquinovose, with major accumulations of sulfopropandiol (2,3-dihydroxypropanesulfonate) but no detectable release of sulfate. Later, when sulfoquinovose was exhausted and growth was very slow, sulfopropandiol disappeared and inorganic sulfate and small amounts of sulfolactate (2-hydroxy-3-sulfopropionate) were formed. In Agrobacterium sp. strain ABR2, growth and sulfoquinovose disappearance were again coincident, though slower than that in Klebsiella sp. Release of sulfate was still late but was faster than that in Klebsiella sp., and no metabolites were detected by 13 C-NMR. Extracts of both strains grown on sulfoquinovose contained phosphofructokinase activities that remained unchanged when fructose 6-phosphate was replaced in the assay mixture with either glucose 6-phosphate or sulfoquinovose. The results were consistent with the operation of the Embden-Meyerhoff-Parnas (glycolysis) pathway for catabolism of sulfoquinovose. Extracts of Klebsiella but not Agrobacterium also contained an NAD ؉ -dependent sulfoquinovose dehydrogenase activity, indicating that the Entner-Doudoroff pathway might also contribute to catabolism of sulfoquinovose.