Identification of the active site of phosphoribosyl-dephospho-coenzyme A transferase and relationship of the enzyme to an ancient class of nucleotidyltransferases.
Malonate decarboxylase from Klebsiella pneumoniae contains an acyl carrier protein (MdcC) to which a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group is attached via phosphodiester linkage to serine 25. We have shown in the preceding paper in this issue that the formation of this phosphodiester bond is catalyzed by a phosphoribosyl-dephospho-coenzyme A transferase MdcG with the substrate 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA that is synthesized from ATP and dephospho-coenzyme A by the triphosphoribosyl transferase MdcB. The reaction catalyzed by MdcG is related to nucleotidyltransfer reactions, and the enzyme indeed catalyzes unphysiological nucleotidyltransfer, e.g., adenylyltransfer from ATP to apo acyl carrier protein (ACP). These unspecific side reactions are favored at high Mg(2+) concentrations. A sequence motif including D134 and D136 of MdcG is a signature of all nucleotidyltransferases. It is known from the well-characterized mammalian DNA polymerase beta that this motif is at the active site of the enzyme. Site-directed mutagenesis of D134 and/or D136 of MdcG to alanine abolished the transfer of the prosthetic group to apo ACP, but the binding of triphosphoribosyl-dephospho-CoA to MdcG was not affected. Evidence is presented that similar to MdcG, MadK encoded by the malonate decarboxylase operon of Malonomonas rubra and CitX from the operon encoding citrate lyase in Escherichia coli are phosphoribosyl-dephospho-CoA transferases catalyzing the attachment of the phosphoribosyl-dephospho-CoA prosthetic group to their specific apo ACPs.
Hoenke
S
S
Institut für Mikrobiologie der Eidgenössischen Technischen Hochschule Zürich, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland.
Schmid
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M
Dimroth
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P
D000213:Acyl Carrier Protein / Q000096:biosynthesis / Q000378:metabolism; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000096:biosynthesis; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism; D002384:Catalysis; D003065:Coenzyme A / Q000378:metabolism*; D020564:Deltaproteobacteria / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D005810:Multigene Family; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000213:Acyl Carrier Protein; D001059:Apoproteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C015787:dephosphocoenzyme A; D000255:Adenosine Triphosphate; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; C494968:fabD protein, E coli; D009713:Nucleotidyltransferases; D002262:Carboxy-Lyases; C071852:malonate decarboxylase; D007652:Oxo-Acid-Lyases; C019909:citrate (pro-3S)-lyase; D054889:Fatty Acid Synthase, Type II; D008274:Magnesium; D003065:Coenzyme A