Identification of Active Site Catalytic Residue in Isovaleryl-CoA Dehydrogenase from Paracoccus denitrificans Pd1222

The gene of Pden_3633 in Paracoccus denitrificans Pd1222, Isovaleryl-CoA dehydrogenase gene (IVDH), was synthesized, cloned, expressed into E. coli BL21 (DE3) using pET24d vector, and purified as N-terminal Strep-Tagged enzyme (Karim and Hashim, 2016a; Karim and Hashim; 2016b). In current study, a Site-directed mutagenesis was used to identify the active site catalytic residue of this synthetic Sterp-Tag IVDH enzyme. Amino acid alignment showed that the E246 is the predicted active site catalytic residue. To substantiate the role of E246 as a catalytic residue, a mutant E246Q IVDH was constructed. Spectral properties of the mutant IVDH indicated that it was obtained as an apoprotein. Therefore, the protein was full reconstituted by incubation with flavin adenine dinucleotide (FAD) at a ratio 1: 20% (IVDH: FAD) molar excess. The results revealed that the reconstituted E246Q IVDH had no activity for isovaleryl-CoA. Furthermore, its UV/visible spectrum resulted from titration with isovaleryl-CoA did not induce quenching of the absorption at 364 and 440 nm regions or arise a new absorption at 598 nm as wild type did. Confirming that the mutant IVDH was unable to form charge transfer complex as a result of altering E246 and the later is the active site catalytic residue of P. denitrificans IVDH