Identification of amino acid changes affecting yeast uroporphyrinogen decarboxylase activity by sequence analysis of hem12 mutant alleles

Author:

Chelstowska A1,Zoladek T1,Garey J2,Kushner J2,Rytka J1,Labbe-Bois R3

Affiliation:

1. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul Rakowiecka 36, 02-532 Warsaw, Poland

2. Division of Hematology and Oncology, University of Utah, School of Medicine, Salt Lake City, UT 84132, U.S.A.

3. Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod, Universite Paris 7, 2 Place Jussieu, 75251 Paris Cédex 05, France

Abstract

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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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