In vitro characterization of salmochelin and enterobactin trilactone hydrolases IroD, IroE, and Fes.
The iroA locus encodes five genes (iroB, iroC, iroD, iroE, iroN) that are found in pathogenic Salmonella and Escherichia coli strains. We recently reported that IroB is an enterobactin (Ent) C-glucosyltransferase, converting the siderophore into mono-, di-, and triglucosyl enterobactins (MGE, DGE, and TGE, respectively). Here, we report the characterization of IroD and IroE as esterases for the apo and Fe(3+)-bound forms of Ent, MGE, DGE, and TGE, and we compare their activities with those of Fes, the previously characterized enterobactin esterase. IroD hydrolyzes both apo and Fe(3+)-bound siderophores distributively to generate DHB-Ser and/or Glc-DHB-Ser, with higher catalytic efficiencies (k(cat)/K(m)) on Fe(3+)-bound forms, suggesting that IroD is the ferric MGE/DGE esterase responsible for cytoplasmic iron release. Similarly, Fes hydrolyzes ferric Ent more efficiently than apo Ent, confirming Fes is the ferric Ent esterase responsible for Fe(3+) release from ferric Ent. Although each enzyme exhibits lower k(cat)'s processing ferric siderophores, dramatic decreases in K(m)'s for ferric siderophores result in increased catalytic efficiencies. The inability of Fes to efficiently hydrolyze ferric MGE, ferric DGE, or ferric TGE explains the requirement for IroD in the iroA cluster. IroE, in contrast, prefers apo siderophores as substrates and tends to hydrolyze the trilactone just once to produce linearized trimers. These data and the periplasmic location of IroE suggest that it hydrolyzes apo enterobactins while they are being exported. IroD hydrolyzes apo MGE (and DGE) regioselectively to give a single linear trimer product and a single linear dimer product as determined by NMR.
Lin
Hening
H
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Fischbach
Michael A
MA
Liu
David R
DR
Walsh
Christopher T
CT
D002384:Catalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006868:Hydrolysis; D007700:Kinetics; D012475:Salmonella / Q000201:enzymology / Q000235:genetics
D006867:Hydrolases