Phosphopantothenoylcysteine synthetase (PPCS; coaB) is responsible for the installation of the essential cystamine moiety of phosphopantetheine by catalyzing the nucleotide-dependent condensation of L-cysteine and 4'-phosphopantothenate to form phosphopantothenoylcysteine.  A novel Type 3, monofunctional PPCS has been recently cloned and identified in Enterococcus faecalis and Streptococcus pneumoniae.  Herein, site directed, saturation mutagenesis of S. pneumoniae PPCS was employed to elucidate structure/function relationships of individual residues highly conserved across bacterial PPCSs.  A unique in vivo evaluation system was established consisting of plasmid-based expression of S. pneumoniae PPCS mutants in an E. coli MG1655 PPCS knockout cell line (?panC,?coaBC::KanR) to rapidly determine active mutants.  The requisite nature of conserved basic and acidic active site residues in Type 3 PPCS has been established and active mutants kinetically characterized.

Function of conserved residues in monofunctional phosphopantothenoylcysteine synthetase

244th National Meeting (2012)

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