MOLECULAR ANALYSIS OF 2 PYRUVATE-DEHYDROGENASE KINASES FROM MAIZE

Two maize cDNAs were isolated and sequenced that had open reading fram es with approximately 37% amino acid identity to mammalian pyruvate de hydrogenase kinases. Both maize kinase sequences contain the five doma ins with conserved signature residues typical of procaryotic two-compo nent histidine kinases. Sequence comparisons identified six other high ly conserved motifs that are proposed to be specific to pyruvate dehyd rogenase kinases. In addition, specific Trp and Cys residues are also invariant in these sequences. The maize cDNAs are 1332 (PDK1) and 1602 (PDK2) nucleotides in length, encoding polypeptides with calculated m olecular masses of 38,867 and 41,327 Da that share 77% amino acid iden tity, Reverse transcriptase-polymerase chain reaction analysis with ol igonucleotide-specific primers revealed a differential expression patt ern for the two isoforms, PDK1 and PDK2 were expressed in Escherichia coli with N-terminal His(6) tags to facilitate purification. The recom binant proteins migrated at 44 and 48 kDa, respectively, during SDS-po lyacrylamide gel electrophoresis. Anti-PDK1 antibodies immunoprecipita ted 75% of pyruvate dehydrogenase kinase activity from a maize mitocho ndrial matrix fraction, and recognized a matrix protein of 43 kDa. Rec ombinant PDK2, expressed as a fusion with the maltose-binding protein, inactivated kinase-depleted maize pyruvate dehydrogenase complex when incubated with MgATP, coincident with incorporation of P-32 from [gam ma-P-32]ATP into the alpha subunit of pyruvate dehydrogenase.