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Nucleotide sequence for yeast dihydrolipoamide dehydrogenase.
K S Browning1, D J Uhlinger, L J Reed
1Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
Summary
Researchers identified the gene for yeast dihydrolipoamide dehydrogenase, a key enzyme in the pyruvate dehydrogenase complex. This finding reveals significant amino acid sequence conservation with bacterial and mammalian counterparts, particularly at the active site.
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- The pyruvate dehydrogenase complex is crucial for cellular energy metabolism.
- Dihydrolipoamide dehydrogenase (EC 1.8.1.4) is a key component of this complex.
- Understanding the structure and function of this enzyme is vital for metabolic research.
Purpose of the Study:
- To isolate and characterize the cDNA encoding yeast dihydrolipoamide dehydrogenase.
- To analyze the deduced amino acid sequence and compare it with homologous proteins.
- To investigate evolutionary conservation within the dihydrolipoamide dehydrogenase family.
Main Methods:
- Screening of a lambda gt11 yeast cDNA library using specific rabbit antiserum.
- Hybridization with an oligonucleotide probe based on the N-terminal amino acid sequence.
- Computer-assisted analysis of the isolated cDNA sequence and deduced protein structure.
Main Results:
- A 2.1-kilobase cDNA insert encoding dihydrolipoamide dehydrogenase was isolated.
- The deduced protein sequence includes a 21-residue signal peptide and a 478-residue mature enzyme (Mr = 51,558).
- Yeast dihydrolipoamide dehydrogenase exhibits 41% amino acid identity with its Escherichia coli counterpart, with conserved active site regions across species.
Conclusions:
- The study successfully identified and characterized the yeast dihydrolipoamide dehydrogenase cDNA.
- Significant sequence conservation highlights the functional importance of the active site across diverse organisms.
- This research provides a foundation for further studies on the enzyme's structure-function relationship and evolutionary history.