On of a residue corresponding to Arg16CaiB by lysine (Lys13TBEA6) in V. paradoxus strain TBEA6

On of a residue corresponding to Arg16CaiB by lysine (Lys13TBEA6) in V. paradoxus strain TBEA6 and an extra glutamine residue (Gln196TBEA6) among Leu195TBEA6 (corresponding to Leu184CaiB) and His197TBEA6 (corresponding to His185CaiB). CDK11 list secondary structure analyses. The amino acid sequences of ActTBEA6 and its orthologues were subjected to secondary structure prediction by the Jpred server (44) (see Fig. S2 in the supplemental material). On account of their readily available solved crystal structures, formyl-CoA:oxalate CoA-transferase from E. coli (YfdW) (27, 28), its orthologue Frc from Oxalobacter formigenes (20, 26), and crotonobetainyl-CoA:carnitine CoA-transferase from E. coli (CaiB) (29, 30) as members on the CoA-transferase III family were included for comparison. As shown in Fig. S2, the amino acid sequences of ActTBEA6, ActDPN7, and ActLB400 (YP_553419.1) are truncated by about 13 to 15 amino acid residues in comparison to all other included sequences. Cloning of the putative acyl-CoA-transferase gene actTBEA6 in to the vector pET22b( ), overexpression in E. coli Lemo21(DE3), and purification and characterization with the translational solution. Based on nucleotide sequence information (GenBank accession no. ACC69030.two), native ActTBEA6 has a calculated molecular mass of 43.322 kDa (isotopically average), consists of 398 amino acids, and has a calculated pI of five.46. In this study, the putative act gene of V. paradoxus strain TBEA6 was heterologously expressed as a His6-tagged protein using the T7promoter/polymerase-based expression vector pET22b( ) and E. coli Lemo21(DE3) because the host strain. For this, the protein was equipped with an extra C-terminal His6 tag plus two vectorencoded amino acids (leucine and glutamate) and an N-terminal pelB signal sequence (22 amino acids plus 17 amino acids in between pelB plus the commence of act) for potential periplasmatic localization (see Materials and Techniques) (see Fig. S1 within the supplemental material). Consequently, the heterologously expressed protein consisted of 445 amino acids, and it exhibited a theoretical molecular mass of 48.372 Da (isotopically average) and a theoretical pI of five.65. The overproduced enzyme was purified by immobilized metal Glucosylceramide Synthase (GCS) Compound chelate affinity chromatography to electrophoretic homogeneity (Fig. four). Afterwards, ActTBEA6 was applied to analytical size exclusion chromatography. It revealed an apparent molecular mass of 96 3 kDa. This corresponds to a homodimer on the protein using a theoretical molecular mass of 96.7 kDa, including the His6 tag along with the additional 39 amino acid residues with the Nterminal pelB signal sequence. The UV-visible spectrum (jb.asm.orgJournal of BacteriologySuccinyl-CoA:3-Sulfinopropionate CoA-TransferaseFIG four Purification of ActTBEA6 by affinity chromatography as revealed by SDS-PAGE. Lane 1, crude extract of cells; lane M, molecular mass marker; lane 2, soluble fraction following centrifugation; lane 3, elution fraction after Ni-NTA affinity chromatography column; lane 4, pooled fractions recovered following Superdex 200 HR size exclusion chromatography. Forty micrograms of protein was applied in lanes 1 and two. Lanes 3 and four have been loaded with five g protein. The SDS gel was stained with Coomassie brilliant blue R.to 800 nm) of purified ActTBEA6 showed a single peak at 280 nm, which indicates the absence of any chromophoric cofactor. Act enzyme activity assays applying the heterologously expressed and purified protein. (i) Initial identification of an acceptable CoA-donor for any.