Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii.
Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either. Kinetic analysis of the mutants revealed that Lys-170 and His-206 play a critical role for ATP binding and the formation of malonyl-AMP, whereas Arg-168 is critical for formation of malonyl-CoA and specificity for malonyl-AMP. Molecular modelling based on the crystal structures of luciferase and gramicidin S synthetase 1 provided MCS structure which could fully explain all these biochemical data even though the MCS model was generated by comparative modelling.
An
J H
JH
Department of Biochemistry, College of Science, Bioproducts Research Centre, Yonsei University, Seoul 120-749, Korea.
Lee
G Y
GY
Jung
J W
JW
Lee
W
W
Kim
Y S
YS
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C021533:malonyl-CoA synthetase