Preferential hydrolysis of aberrant intermediates by the type II thioesterase in Escherichia coli nonribosomal enterobactin synthesis: substrate specificities and mutagenic studies on the active-site residues.
The type II thioesterase EntH is a hotdog fold protein required for optimal nonribosomal biosynthesis of enterobactin in Escherichia coli. Its proposed proofreading activity in the biosynthesis is confirmed by its efficient restoration of enterobactin synthesis blocked in vitro by analogs of the cognate precursor 2,3-dihydroxybenzoate. Steady-state kinetic studies show that EntH recognizes the phosphopantetheine group and the pattern of hydroxylation in the aryl moiety of its thioester substrates. Remarkably, it is able to distinguish aberrant intermediates from the normal one in the enterobactin assembly line by demonstrating at least 10-fold higher catalytic efficiency toward thioesters derived from aberrant aryl precursors without a para-hydroxyl group, such as salicylate. By structural comparison and site-directed mutagenesis, the thioesterase is found to possess an active site closely resembling that of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. strain SU and to involve an acidic residue (glutamate-63) as the catalytic base or nucleophile like all other hotdog thioesterases. In addition, the EntH specificities toward the substrate hydroxylation pattern are found to depend on the active-site histidine-54, threonine-64, serine-67, and methionine-68 with the selectivity significantly reduced or even reversed when they are individually replaced by alanine. These residues are likely responsible for differential interaction of the enzyme with the substrates which leads to distinction between the normal and aberrant precursors in the enterobactin assembly line. These results show that the type II thioesterase evolves its distinctive ability to recognize the aberrant intermediates from the versatile catalytic platform of hotdog proteins and suggests an active search mechanism for type II thioesterases in nonribosomal peptide synthesis.
Guo
Zu-Feng
ZF
Department of Chemistry, Center for Cancer Research, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
Sun
Yueru
Y
Zheng
Suilan
S
Guo
Zhihong
Z
D000596:Amino Acids / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004758:Enterobactin / Q000096:biosynthesis*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D064429:Fatty Acid Synthases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D062385:Hydroxybenzoates / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D029968:Escherichia coli Proteins; D062385:Hydroxybenzoates; D050505:Mutant Proteins; D004758:Enterobactin; D064429:Fatty Acid Synthases; C527558:EntH protein, E coli; D013869:Thiolester Hydrolases; C022738:thioesterase II