Submitted to: Journal of Biological Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/2/1999
Publication Date: N/A
Citation: Interpretive Summary: Respiration can be thought of as the use of energy by living cells to do work. Important components of this work include growth and reproduction. Respiration must be carefully controlled or energy will be wasted. Wasted energy would decrease crop yields and reduce agricultural productivity. The control of respiration in plant cells is not yet fully understood. The genetic material for an important control step in respiration was isolated from corn and studied. Comparisons were made with similar genetic material from animals and fungi in order to identify which parts might be important in control of respiration. A method was developed to prepare large amounts of the plant products that had full biological activity. Being able to produce large amounts of material will allow detailed analysis of the structures. This information will be important to researchers in their attempts to increase agricultural productivity by altering the control of plant cell respiration and to other plant scientists who will try to design more efficient crop plants through either classical breeding or biotechnology.
Technical Abstract: The dihydrolipoamide acetyltransferase subunit of the maize mitochondrial pyruvate dehydrogenase complex was previously postulated to contain a single lipoyl domain based upon a molecular mass of 52 kDa and the N-terminal deduced amino acid sequence (Thelen et al., 1998a). Using the deduced amino acid sequence, a maize cDNA was identified from an Expressed Sequence Tag database. The primary amino acid sequence was 30-37% identical to other dihydrolipoamide acetyltransferases and contained a single lipoyl domain. Since the only other plant dihydrolipoamide acetyltransferase sequence contains two lipoyl domains and is only 30% identical (Guan et al., 1995), the activity and assembly properties of this recombinant protein were investigated. The recombinant protein, expressed in E. coli as the mature polypeptide without any fusion tags, was purified to a specific activity of 191 units/mg protein, using conventional methods. The purified recombinant protein exhibited acetyltransferase activity using two different methods, and assembled into a complex of approximately 2.7 mDa based upon size exclusion chromatography. The purified recombinant complex assembled into a 25 nm pentagonal dodecahedron core with icosahedral symmetry as visualized by negative-staining transmission electron microscopy.