TY - JOUR
T1 - Stoichiometry and specificity of in vitro phosphopantetheinylation and aminoacylation of the valine-activating module of surfactin synthetase
AU - Weinreb, Paul H.
AU - Quadri, Luis E.N.
AU - Walsh, Christopher T.
AU - Zuber, Peter
PY - 1998/2/10
Y1 - 1998/2/10
N2 - 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 SrfB 1 fragment that contains four domains putatively involved in activation (adenylation domain), autoaminoacylation (peptidyl carder 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.
AB - 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 SrfB 1 fragment that contains four domains putatively involved in activation (adenylation domain), autoaminoacylation (peptidyl carder 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.
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U2 - 10.1021/bi9719859
DO - 10.1021/bi9719859
M3 - Article
C2 - 9484228
AN - SCOPUS:0032502031
SN - 0006-2960
VL - 37
SP - 1575
EP - 1584
JO - Biochemistry
JF - Biochemistry
IS - 6
ER -