Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate.
In Escherichia coli, the siderophore molecule enterobactin is synthesized in response to iron deprivation by formation of an amide bond between 2,3-dihydroxybenzoate (2,3-DHB) and l-serine and formation of ester linkages between three such N-acylated serine residues. We show that EntB, previously described as the isochorismate lyase required for production of 2,3-DHB, is a bifunctional protein that also serves as an aryl carrier protein (ArCP) with a role in enterobactin assembly. EntB is phosphopantetheinylated near the C terminus in a reaction catalyzed by EntD with a kcat of 5 min-1 and a Km for apo-EntB of 6.5 microM. This holo-EntB is then acylated with 2,3-DHB in a reaction catalyzed by EntE, previously described as the 2,3-DHB-AMP ligase, with a kcat of 100 min-1 and a Km of <<1 microM for holo-EntB. The N-terminal 187 amino acids of EntB (isochorismate lyase domain) are not needed for reaction of EntB with either EntD or EntE as demonstrated by the equivalent catalytic efficiencies of the full-length EntB (residues 1-285) and the C-terminal EntB ArCP domain (residues 188-285) as substrates for both EntD and EntE.
Gehring
A M
AM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Bradley
K A
KA
Walsh
C T
CT
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004758:Enterobactin / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D013058:Mass Spectrometry; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D010204:Pantetheine / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012459:Salicylates / Q000378:metabolism; D020156:Salicylic Acid; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D014166:Transferases / Q000378:metabolism*
D029968:Escherichia coli Proteins; D062385:Hydroxybenzoates; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012459:Salicylates; D004758:Enterobactin; D012694:Serine; D010204:Pantetheine; C009135:2,3-dihydroxybenzoic acid; D000255:Adenosine Triphosphate; D014166:Transferases; D006867:Hydrolases; C064012:isochorismatase; D008025:Ligases; C061746:2,3-dihydroxybenzoate-AMP ligase, E coli; C003129:4'-phosphopantetheine; D020156:Salicylic Acid