Stoichiometry and specificity of in vitro phosphopantetheinylation and aminoacylation of the valine-activating module of surfactin synthetase.

Abstract

Surfactin synthetase is the enzyme responsible for biosynthesis of the lipoheptapeptide antibiotic surfactin by Bacillus subtilis. Fragments of SrfB1, the L-valine-activating module of the second subunit of surfactin synthetase, were overproduced in Escherichia coli. In addition to a 143-kDa SrfB1 fragment that contains four domains putatively involved in activation (adenylation domain), autoaminoacylation (peptidyl carrier protein (PCP) domain), and peptide bond formation (two condensation domains), subfragments comprising two domains (104-kDa condensation-adenylation and 73-kDa adenylation-PCP), and one domain (18-kDa PCP) were also overproduced in and purified from E. coli as N-terminal hexahistidine fusion proteins. Incubation of these domains with pure Sfp, a phosphopantetheinyl transferase (PPTase) from B. subtilis, and CoA allowed quantitation of posttranslational phosphopantetheinylation of Ser999 by mass spectrometry for the 18-kDa PCP fragment and by radioassay using cosubstrate [3H] pantetheinyl-coenzyme A for all PCP-containing constructs. The phosphopantetheine stoichiometry correlated with the subsequent mole fractions of [14C] valyl groups that could be covalently transferred to these holo-PCP domains. In turn, the catalytic efficiency of intramolecular aminoacylation of the 143-kDa fragment could be compared with the reaction "in trans" between adenylation and PCP fragments of SrfB1. The corresponding holo-PCP domain of the next module, SrfB2, was not detectably aminoacylated by SrfB1, indicative of protein-protein recognition between adenylation and cognate PCP domains. These results should permit future exploration of the timing and specificity of peptide bond formation by this class of biosynthetic enzymes.