Identification of amino acid changes affecting yeast uroporphyrinogen decarboxylase activity by sequence analysis of hem12 mutant alleles.
The molecular basis of the uroporphyrinogen decarboxylase defect in eleven yeast 'uroporphyric' mutants was investigated. Uroporphyrinogen decarboxylase, an enzyme of the haem-biosynthetic pathway, catalyses the decarboxylation of uroporphyrinogen to coproporphyrinogen and is encoded by the HEM12 gene in the yeast Saccharomyces cerevisiae. The mutations were identified by sequencing the mutant hem12 alleles amplified in vitro from genomic DNA extracted from the mutant strains. Four mutations leading to the absence of enzyme protein were found: one mutation caused the substitution of the translation initiator Met to Ile, a two-base deletion created a frameshift at codon 247 and two nonsense mutations were found at codons 50 and 263. Four different point mutations were identified in seven 'leaky' mutants with residual modified uroporphyrinogen decarboxylase activity; each of three mutations was found in two independently isolated mutants. The nucleotide transitions resulted in the amino acid substitutions Ser-59 to Phe, Thr-62 to Ile, Leu-107 to Ser, or Ser-215 to Asn, all located in or near highly conserved regions. The results suggest that there is a single active centre in uroporphyrinogen decarboxylase, the geometry of which is affected in the mutant enzymes.
Chelstowska
A
A
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
Zoladek
T
T
Garey
J
J
Kushner
J
J
Rytka
J
J
Labbe-Bois
R
R
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D010641:Phenotype; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013329:Structure-Activity Relationship; D014575:Uroporphyrinogen Decarboxylase / Q000235:genetics* / Q000378:metabolism
D014575:Uroporphyrinogen Decarboxylase