Cloning of two isozymes of Trichoderma koningii glyceraldehyde-3-phosphate dehydrogenase with different sensitivity to koningic acid.
Koningic acid inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by binding to the SH group in the active center. The fungus Trichoderma koningii, the producer of koningic acid, contains two GAPDH isozymes (GAPDHs I and II). GAPDH I is inhibited 50% by 1.1.10(-3) M koningic acid, while GAPDH II is inhibited 50% at 6.8 x 10(-6) M. cDNAs of the two isozymes were cloned from T. koningii and their nucleotide sequences were determined. The sequence of coding region and codon usage in both clones were compared with each other and with those of the gene for Aspergillus nidulans GAPDH (enzyme activity is inhibited 50% by 2.7 x 10(-7) M koningic acid). Results indicated that GAPDH II is more closely related to A. nidulans GAPDH than GAPDH I. All essential amino acid residues, except 174 and 181, which are implicated in catalysis and binding of NAD and substrates, were conserved among A. nidulans GAPDH and GAPDHs I and II. Residues 174 and 181 are threonine in both A. nidulans GAPDH and GAPDH II, but alanine and serine, respectively, in GAPDH I. The side-chain of alanine-174 in GAPDH I can not replace threonine-174 functionally as threonine-174 side-chain forms a hydrogen bond with the catalytically essential histidine-176.
Watanabe
H
H
Department of Applied Biological Science, Tokyo Noko University, Japan.
Hasumi
K
K
Fukushima
Y
Y
Sakai
K
K
Endo
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012717:Sesquiterpenes / Q000494:pharmacology; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics*
D004271:DNA, Fungal; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C027285:heptelidic acid; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases