CHARACTERIZATION OF WILD-TYPE AND AN ACTIVE-SITE MUTANT IN ESCHERICHIA-COLI OF SHORT-CHAIN ACYL-COA DEHYDROGENASE FROM MEGASPHAERA-ELSDENII

The objective of this work is to determine the molecular mechanism and regulation of short-chain acyl-CoA dehydrogenase (SCAD) from Megaspha era elsdenii. To achieve this, the gene coding for SCAD from M. elsden ii was cloned and sequenced. Site-directed mutagenesis was then used t o identify an amino acid residue that is required for the proposed mec hanism. To clone the gene, the amino acid sequence of the 50 N-termina l residues of SCAD from M. elsdenii was determined. This sequence info rmation was utilized to synthesize two sets of mixed oligonucleotide p rimers 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 i nto Escherichia coli. The gene encoding M. elsdenii SCAD was identifie d from this library, sequenced, and expressed. The cloned SCAD gene co ntained an open reading frame which revealed a high degree of sequence identity with an open reading frame protein sequence of the human SCA D 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 account ed for 35% of the cytosolic protein in the Escherichia coli crude extr act. The expressed protein had similar activity, redox potential prope rties, and nearly identical amino acid composition to native M. elsden ii SCAD. In addition, a site-directed Glu367Gln mutant of SCAD express ed from a pUC119 vector was shown to have minimal reductive and oxidat ive 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 mut ants that will enable us to characterize the mechanism and thermodynam ic regulation of M. elsdenii SCAD.