Leucine biosynthesis in fungi: entering metabolism through the back door

Abstract

After exploring evolutionary aspects of branched-chain amino acid biosynthesis, the review focuses on the extended leucine biosynthetic pathway as it operates in Saccharomyces cerevisiae. First, the genes and enzymes specific for the leucine pathway are considered: LEU4 and LEU9 (encoding the alpha-isopropylmalate synthase isoenzymes), LEU1 (isopropylmalate isomerase), and LEU2 (beta-isopropylmalate dehydrogenase). Emphasis is given to the unusual distribution of the branched-chain amino acid pathway enzymes between mitochondrial matrix and cytosol, on the newly defined role of Leu5p, and on regulatory mechanisms governing gene expression and enzyme activity, including new evidence for the metabolic importance of the regulation of alpha-isopropylmalate synthase by coenzyme A. Next, structure-function relationships of the transcriptional regulator Leu3p are addressed, defining its dual role as activator and repressor and discussing evidence in support of the self-masking model. Recent data pointing at a more extended Leu3p regulon are discussed. An overview of the layered controls of the extended leucine pathway is provided that includes a description of the newly recognized roles of Ilv5p and Bat1p in maintaining mitochondrial integrity. Finally, branched-chain amino acid biosynthesis and its regulation in other fungi are summarized, the question of leucine as metabolic signal is addressed, and possible directions of future research in this area are outlined.

FIG. 1.

Compartmentation of the branched-chain amino acid biosynthetic pathways of S. cerevisiae. The boxed characters refer to the genes involved directly or indirectly in the pathways. Their protein products are as follows: ILV1, threonine deaminase; ILV2, acetohydroxy acid synthase catalytic subunit; ILV5, acetohydroxy acid reductoisomerase; ILV3, dihydroxy acid dehydratase; BAT1, mitochondrial branched-chain amino acid aminotransferase; BAT2, cytosolic branched-chain amino acid aminotransferase; LEU4l, α-isopropylmalate synthase I, long form; LEU4s, α-isopropylmalate synthase I, short form; LEU9, α-isopropylmalate synthase II; X, hypothetical α-isopropylmalate transporter; LEU1, isopropylmalate isomerase; LEU2, β-isopropylmalate dehydrogenase; LEU5, protein necessary for the accumulation of CoA within the mitochondria; presumably an importer of CoA or precursor thereof; ATM1, ABC transporter involved in exporting Fe-S clusters to the cytosol; the implied interaction between Bat1p and Atmp is hypothetical (58, 66). Abbreviations: KB, α-ketobutyrate; AALD, active acetaldehyde; PYR, pyruvate; AL, acetolactate; AHB, α-aceto-α-hydroxybutyrate; DHIV, α,β-dihydroxyisovalerate; DHMV, α,β-dihydroxy-β-methylvalerate; KIV, α-ketoisovalerate; KMV, α-keto-β-methylvalerate; α-IPM, α-isopropylmalate; β-IPM, β-isopropylmalate; KIC, α-ketoisocaproate; Fe/S, iron-sulfur cluster. Not shown for reasons of clarity: transport of leucine and/or KIC back into the mitochondria.

FIG. 2.

Alignment of the R-regions (boxed) of known α-isopropylmalate synthase sequences. Amino acid sequence identities are indicated by bold letters, similarities are indicated by asterisks. Abbreviations and references: ScI, S. cerevisiae α-isopropylmalate synthase I (; the amino acid positions shown at the top refer to this sequence); Sc II, S. cerevisiae α-isopropylmalate synthase II (107); Cg, Corynebacterium glutamicum (82); Ba, Buchnera aphidicola (16); Hi, Haemophilus influenzae (34); Mj, Methanococcus jannaschii (18); St, Salmonella enterica serovar Typhimurium (90); Ll, Lactococcus lactis (38); Ta, Thermus aquaticus (GenBank accession number U52907). Reprinted from reference with permission of the copyright owner.

FIG. 3.

Regions of interest along the primary structure of Leu3p of S. cerevisiae.^aDNA binding region, residues 37 to 67, containing the cysteine motif for Zn(II)₂Cys₆ cluster formation;^bMHR, residues 300 to 380;^cthree clusters of residues (604 to 611, 643 to 664, and 738 to 741) potentially involved in activation domain masking;^dregion dispensable for all known functions, residues 774 to 854;^etranscriptional activation domain, residues 857 to 875; N, N terminus; C, C terminus. Note that the 11 C-terminal residues (residues 876 to 886), while not needed for the activation function, are essential for the masking process. See the text for further details.

FIG. 4.

Major regulatory mechanisms impacting the extended leucine pathway of S. cerevisiae. For abbreviations of intermediates and the protein products of the genes shown, see the legend to Fig. 1. See the text for further explanation.