Purification of Active E1α2β2 of Pseudomonas Putida Branched‐Chain‐Oxoacid Dehydrogenase

Active E1 component of Pseudomonas putida branched-chain-oxoacid dehydrogenase was purified from P. putida strains carrying pJRS84 which contains bkdR (encoding the transcriptional activator) and bkdA1 and bkdA2 (encoding the α and β) subunits). Expression was inducible, however, 45– 39– and 37-kDa proteins were produced instead of the expected 45-kDa and 37-kDa proteins. The 45-kDa protein was identified as E1α and the 37-kDa and 39-kDa proteins were identified as separate translational products of bkdA2 by their N-terminal sequences. The N-terminal amino acid of the 39-kDa protein was leucine instead of methionine. The 45–, 39– and 37-kDa proteins were also produced in wild-type P. putida. Translation of bkdA1 and bkdA2 from an Escherichia coli expression plasmid produced only 45-kDa and 39-kDa proteins, with N-terminal methionine on the 39-kDa protein. The insertion of guanine residues 5′ to the first ATG of bkdA2 did not affect expression of E1β in P. putida including the N-terminal leucine which appears to eliminate the possibility of ribosome jumping. The Z-average molecular mass of the El component was determined by sedimentation equilibrium to be 172±9 kDa compared to a calculated value of 166 kDa for the heterotetramer and a Stokes radius of 5.1 nm. E1α Ser313, which is homologous to the phosphorylated residue of rat liver E1α, was converted to alanine resulting in about a twofold increase in Km, but no change in Kcat. S315A and S319A mutations had no effect on Km or Kcat, indicating that these residues do not play a major part in catalysis of E1α2β2.