CHARACTERIZATION OF WILD-TYPE AND AN ACTIVE-SITE MUTANT IN ESCHERICHIA-COLI OF SHORT-CHAIN ACYL-COA DEHYDROGENASE FROM MEGASPHAERA-ELSDENII
CHARACTERIZATION OF WILD-TYPE AND AN ACTIVE-SITE MUTANT IN ESCHERICHIA-COLI OF SHORT-CHAIN ACYL-COA DEHYDROGENASE FROM MEGASPHAERA-ELSDENII
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DOI：
10.1021/bi00091a026
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发表时间：
1993-10-12
期刊：
影响因子：
2.9
通讯作者：
STANKOVICH, MT
中科院分区：
文献类型：
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作者：
BECKER, DF;FUCHS, JA;STANKOVICH, MT
本研究旨在探讨埃氏巨球酵母短链酰基辅酶A脱氢酶（SCAD）的分子机制及其调控。为了实现这一点，从M. elsdenii进行克隆和测序。然后使用定点诱变来鉴定所提出的机制所需的氨基酸残基。为了克隆该基因，从M. elsdenii已确定。利用该序列信息合成两套混合寡核苷酸引物，然后用于产生来自M的120-bp特异性探针。通过聚合酶链反应（PCR）方法对elsdenii DNA进行扩增。用120 bp的探针筛选M.将elsdenii基因组DNA文库克隆到大肠杆菌中。编码M.从该文库中鉴定elsdenii SCAD，测序并表达。克隆的SCAD基因包含一个开放阅读框，其与人SCAD和大鼠中链酰基辅酶A脱氢酶（MCAD）的开放阅读框蛋白序列具有高度的序列同一性（在人SCAD和大鼠MCAD的配对比较中，分别有44%和36%的相同残基）。从pUC 119载体表达的重组SCAD占大肠杆菌粗提物中胞浆蛋白的35%。表达的蛋白具有与天然M相似的活性、氧化还原电位特性和几乎相同的氨基酸组成。elsdenii SCAD.此外，从pUC 119载体表达的SCAD的定点Glu 367 Gln突变体显示分别与丁酰-CoA和巴豆酰-CoA具有最小的还原和氧化途径活性。残基Glu 361已被提出通过提取底物α-质子来引发催化。这是第一个定点突变体，将使我们能够表征的机制和热力学调节的M。elsdenii SCAD.
The objective of this work is to determine the molecular mechanism and regulation of short-chain acyl-CoA dehydrogenase (SCAD) from Megasphaera elsdenii. To achieve this, the gene coding for SCAD from M. elsdenii was cloned and sequenced. Site-directed mutagenesis was then used to identify an amino acid residue that is required for the proposed mechanism. To clone the gene, the amino acid sequence of the 50 N-terminal residues of SCAD from M. elsdenii was determined. This sequence information was utilized to synthesize two sets of mixed oligonucleotide primers which were then used to generate a 120-bp specific probe from M. elsdenii DNA by the polymerase chain reaction (PCR) method. The 120-bp probe was used to screen a M. elsdenii genomic DNA library cloned into Escherichia coli. The gene encoding M. elsdenii SCAD was identified from this library, sequenced, and expressed. The cloned SCAD gene contained an open reading frame which revealed a high degree of sequence identity with an open reading frame protein sequence of the human SCAD and the rat medium-chain acyl-CoA dehydrogenase (MCAD) (44% and 36% identical residues in paired comparisons for human SCAD and rat MCAD, respectively). Recombinant SCAD expressed from a pUC119 vector accounted for 35% of the cytosolic protein in the Escherichia coli crude extract. The expressed protein had similar activity, redox potential properties, and nearly identical amino acid composition to native M. elsdenii SCAD. In addition, a site-directed Glu367Gln mutant of SCAD expressed from a pUC119 vector was shown to have minimal reductive and oxidative pathway activity with butyryl-CoA and crotonyl-CoA, respectively. Residue Glu361 has been proposed to initiate catalysis by abstracting the substrate alpha-proton. This is the first of the site-directed mutants that will enable us to characterize the mechanism and thermodynamic regulation of M. elsdenii SCAD.