Molecular characterization of the 4 '-phosphopantothenoylcysteine decarboxylase domain of bacterial Dfp flavoproteins

The NH2-terminal domain of the bacterial flavoprotein Dfp catalyzes the dec arboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantethe ine, a key step in coenzyme A biosynthesis, Dfp proteins, LanD proteins (fo r example EpiD, which is involved in epidermin biosynthesis), and the salt tolerance protein AtHAL3a from Arabidopsis thaliana are homooligomeric flav in-containing Cys decarboxylases (HFCD protein family), The crystal structu re of the peptidyl-cysteine decarboxylase EpiD complexed with a pentapeptid e substrate has recently been determined. The peptide is bound by an NH2-te rminal substrate binding helix, residue Asn(117), which contacts the cystei ne residue of the substrate, and a COOH-terminal substrate recognition clam p, The conserved motif G-G/S-I-A-X-Y-K of the Dfp proteins aligns partly wi th the substrate binding helix of EpiD. Point mutations within this motif r esulted in loss of coenzyme binding (G14S) or in significant decrease of Df p activity (G15A, I16L, A17D, K20N, K20Q), Exchange of Asn(125) of Dfp, whi ch corresponds to Asn(117) of EpiD, and exchange of Cys(158), which is with in the proposed substrate recognition clamp of Dfp, led to inactivity of th e enzyme, Molecular analysis of the conditional lethality of the Escherichi a coli dfp-707 mutant revealed that the single point mutation G11D of Dfp i s related to decreased amounts of soluble Dfp protein at 37 degreesC.