Molecular characterization of the 4'-phosphopantothenoylcysteine decarboxylase domain of bacterial Dfp flavoproteins.
The NH(2)-terminal domain of the bacterial flavoprotein Dfp catalyzes the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine, a key step in coenzyme A biosynthesis. Dfp proteins, LanD proteins (for example EpiD, which is involved in epidermin biosynthesis), and the salt tolerance protein AtHAL3a from Arabidopsis thaliana are homooligomeric flavin-containing Cys decarboxylases (HFCD protein family). The crystal structure of the peptidyl-cysteine decarboxylase EpiD complexed with a pentapeptide substrate has recently been determined. The peptide is bound by an NH(2)-terminal substrate binding helix, residue Asn(117), which contacts the cysteine residue of the substrate, and a COOH-terminal substrate recognition clamp. The conserved motif G-G/S-I-A-X-Y-K of the Dfp proteins aligns partly with the substrate binding helix of EpiD. Point mutations within this motif resulted in loss of coenzyme binding (G14S) or in significant decrease of Dfp activity (G15A, I16L, A17D, K20N, K20Q). Exchange of Asn(125) of Dfp, which corresponds to Asn(117) of EpiD, and exchange of Cys(158), which is within the proposed substrate recognition clamp of Dfp, led to inactivity of the enzyme. Molecular analysis of the conditional lethality of the Escherichia coli dfp-707 mutant revealed that the single point mutation G11D of Dfp is related to decreased amounts of soluble Dfp protein at 37 degrees C.
Kupke
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Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany. Thomas.Kupke@t-online.de
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism*; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D005420:Flavoproteins; D002262:Carboxy-Lyases; C022373:Phosphopantothenoyl-cysteine decarboxylase