4'-phosphopantetheine biosynthesis in Archaea.
Coenzyme A as the principal acyl carrier is required for many synthetic and degradative reactions in intermediary metabolism. It is synthesized in five steps from pantothenate, and recently the CoaA biosynthetic genes of eubacteria, plants, and human were all identified and cloned. In most bacteria, the so-called Dfp proteins catalyze the synthesis of the coenzyme A precursor 4'-phosphopantetheine. Dfp proteins are bifunctional enzymes catalyzing the synthesis of 4'-phosphopantothenoylcysteine (CoaB activity) and its decarboxylation to 4'-phosphopantetheine (CoaC activity). Here, we demonstrate the functional characterization of the CoaB and CoaC domains of an archaebacterial Dfp protein. Both domains of the Methanocaldococcus jannaschii Dfp protein were purified as His tag proteins, and their enzymatic activities were then identified and characterized by site-directed mutagenesis. Although the nucleotide binding motif II of the CoaB domain resembles that of eukaryotic enzymes, Methanocaldococcus CoaB is a CTP- and not an ATP-dependent enzyme, as shown by detection of the 4'-phosphopantothenoyl-CMP intermediate. The proposed 4'-phosphopantothenoylcysteine binding clamp of the Methanocaldococcus CoaC activity differs significantly from those of other characterized CoaC proteins. In particular, the active site cysteine residue, which otherwise is involved in the reduction of an aminoenethiol reaction intermediate, is not present. Moreover, the conserved Asn residue of the PXMNXXMW motif, which contacts the carboxyl group of 4'-phosphopantothenoylcysteine, is exchanged for His.
Kupke
Thomas
T
Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany. Thomas.Kupke@t-online.de
Schwarz
Wolfgang
W
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D017015:Methanococcales / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009097:Multienzyme Complexes; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D019843:Archaeal Proteins; D009097:Multienzyme Complexes; D010204:Pantetheine; D002262:Carboxy-Lyases; D010453:Peptide Synthases; C003129:4'-phosphopantetheine