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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 ...

Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substratespecificity decarboxylase activity.

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.

A cDNA, cRKIN1, encoding a putative homologue of the yeast (Saccharomyces cerevisuiae) SNFl (6), human cells (7), and Xenopus (8) are also present in pea (9) and Arabidopsis (10). All protein kinases that have been characterized in detail contain a number of key residues and conserved regions in the catalytic domain (11) but can be divided into two classes: those that phosphorylate serine/threonine residues and those that phosphorylate tyrosine residues. In the present report, we present the nucleotide sequence of a cDNA, cRKIN1,** isolated from a rye endosperm cDNA library. The cRKIN1-encoded protein contains all the invariant residues and conserved domains characteristic ofeukaryotic protein-serine/threonine kinases. It is particularly similar to the product ofthe SNFJ gene of yeast (Saccharomyces cerevisiae) (12), a protein affecting global regulation of carbon metabolism, and the expression of cRKIN1 in yeast snfl mutants restores SNFJ function.

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.