Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae. HEM12 gene sequence and evidence for two conserved glycines essential for enzymatic activity.
The HEM12 gene from Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase which catalyzes the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis. The gene was isolated by functional complementation of a hem12 mutant. Sequencing revealed that the HEM12 gene encodes a protein of 362 amino acids with a calculated molecular mass of 41,348 Da. The amino acid sequence shares 50% identity with human and rat uroporphyrinogen decarboxylase and shows 40% identity with the N-terminus of an open reading frame described in Synechococcus sp. We determined the sequence of two hem12 mutations which lead to a totally inactive enzyme. They correspond to the amino acid changes Gly33----Asp and Gly300----Asp, located in two evolutionarily conserved regions. Each of these substitutions impairs binding of substrates without affecting the overall conformation of the protein. These results argue that a single active center exists in uroporphyrinogen decarboxylase.
Garey
J R
JR
Department of Biological Sciences, Duquesne University, Pittsburgh, PA 15282.
Labbe-Bois
R
R
Chelstowska
A
A
Rytka
J
J
Harrison
L
L
Kushner
J
J
Labbe
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D004271:DNA, Fungal / Q000235:genetics; D005998:Glycine / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D014575:Uroporphyrinogen Decarboxylase / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D012333:RNA, Messenger; D014575:Uroporphyrinogen Decarboxylase; D005998:Glycine