Characterization of propionate CoA-transferase from Ralstonia eutropha H16.
In this study, a propionate CoA-transferase (H16_A2718; EC 2.8.3.1) from Ralstonia eutropha H16 (Pct(Re)) was characterized in detail. Glu342 was identified as catalytically active amino acid residue via site-directed mutagenesis. Activity of Pct(Re) was irreversibly lost after the treatment with NaBH₄ in the presence of acetyl-CoA as it is shown for all CoA-transferases from class I, thereby confirming the formation of the covalent enzyme-CoA intermediate by Pct(Re). In addition to already known CoA acceptors for Pct Re such as 3-hydroxypropionate, 3-hydroxybutyrate, acrylate, succinate, lactate, butyrate, crotonate and 4-hydroxybutyrate, it was found that glycolate, chloropropionate, acetoacetate, valerate, trans-2,3-pentenoate, isovalerate, hexanoate, octanoate and trans-2,3-octenoate formed also corresponding CoA-thioesters after incubation with acetyl-CoA and Pct(Re). Isobutyrate was found to be preferentially used as CoA acceptor amongst other carboxylates tested in this study. In contrast, no products were detected with acetyl-CoA and formiate, bromopropionate, glycine, pyruvate, 2-hydroxybutyrate, malonate, fumarate, itaconate, β-alanine, γ-aminobutyrate, levulate, glutarate or adipate as potential CoA acceptor. Amongst CoA donors, butyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, isobutyryl-CoA, succinyl-CoA and valeryl-CoA apart from already known propionyl-CoA and acetyl-CoA could also donate CoA to acetate. The highest rate of the reaction was observed with 3-hydroxybutyryl-CoA (2.5 μmol mg⁻¹ min⁻¹). K(m) values for propionyl-CoA, acetyl-CoA, acetate and 3-hydroxybutyrate were 0.3, 0.6, 4.5 and 4.3 mM, respectively. The rather broad substrate range might be a good starting point for enzyme engineering approaches and for the application of Pct(Re) in biotechnological polyester production.
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, 48149, Münster, Germany.
Elena
E
Volodina
Marc
M
Schürmann
Nicole
N
Lindenkamp
Alexander
A
Steinbüchel
D019943:Amino Acid Substitution; D003065:Coenzyme A / Q000378:metabolism*; D015256:Coenzyme A-Transferases / Q000235:genetics / Q000378:metabolism*; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004252:DNA Mutational Analysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D015256:Coenzyme A-Transferases; C031212:acyl-CoA transferase; D003065:Coenzyme A