Asp578 in LEU4p is one of the key residues for leucine feedback inhibition release in sake yeast.
We identified a new mutation, Asp578Tyr, in alpha-isopropylmalate synthase (a LEU4 gene product) that releases leucine feedback inhibition and causes hyperproduction of isoamyl alcohol (i-AmOH) in sake yeast. Spontaneous sake yeast mutants that express resistance to 5,5,5-trifluoro-DL-leucine (TFL) were isolated, and a mutant strain, TFL20, was characterized at the genetic and biochemical levels. An enzyme assay for alpha-isopropylmalate synthase showed that strain TFL20 was released from feedback inhibition by L-leucine. Furthermore, DNA sequencing of the LEU4 gene for a haploid of the mutant TFL20 revealed that aspartic acid in position 578 changes to tyrosine. A comparison of the three-dimensional structures of wild-type LEU4p and mutant LEU4D578Yp by the homology modeling method showed that Asp578 is important for leucine feedback inhibition. We conclude that the mutation from Asp to Tyr in 578 is a novel change causing release from leucine feedback inhibition.
Oba
Takahiro
T
Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Aikawa-machi 1465-5, Kurume, Fukuoka 839-0861. ooba@fitc.pref.fukuoka.jp
Nomiyama
Shuji
S
Hirakawa
Hideki
H
Tashiro
Kosuke
K
Kuhara
Satoru
S
D015079:2-Isopropylmalate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000235:genetics*; D004171:Diploidy; D025141:Drug Resistance, Fungal; D025461:Feedback, Physiological / Q000187:drug effects; D005838:Genotype; D006238:Haploidy; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D009154:Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014443:Tyrosine / Q000235:genetics
D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; C035177:5',5',5'-trifluoroleucine; D014443:Tyrosine; D015079:2-Isopropylmalate Synthase; D007930:Leucine