Identification of amino acid changes affecting yeast uroporphyrinogen decarboxylase activity by sequence analysis of hem12 mutant alleles.
Identification of amino acid changes affecting yeast uroporphyrinogen decarboxylase activity by sequence analysis of hem12 mutant alleles.
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通过 hem12 突变等位基因的序列分析鉴定影响酵母尿卟啉原脱羧酶活性的氨基酸变化。
DOI：
10.1042/bj2880753
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Labbe-Bois,R
中科院分区：
文献类型：
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作者：
Chelstowska,A;Zoladek,T;Garey,J;Kushner,J;Rytka,J;Labbe-Bois,R
对11株酵母‘尿卟啉’突变体的尿卟啉原脱羧酶缺陷的分子基础进行了研究。尿菜红素原脱羧酶是血色素生物合成途径中的一种酶，催化尿菜红素原脱羧为辅酶红素原，由酿酒酵母中的HEM12基因编码。通过对从突变菌株提取的基因组DNA进行体外扩增的突变hem12等位基因的测序来鉴定突变。发现了4个导致酶蛋白缺失的突变：1个突变导致翻译起始子Met被替换为Ile，2个碱基缺失导致247密码子移码，2个无义突变在50和263密码子上发现。在7个具有残留修饰尿卟啉原脱羧酶活性的“泄漏”突变体中发现了四个不同的点突变；三个突变中的每一个都在两个独立分离的突变体中发现。核苷酸转换导致Ser-59到Phe、Thr-62到Ile、Leu-107到Ser或Ser-215到Asn的氨基酸替换，都位于高度保守的区域或附近。结果表明，尿卟啉原脱羧酶只有一个活性中心，其几何构型受突变酶的影响。
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.