Adenylate forming enzymes involved in NRPS-independent siderophore biosynthesis

Abstract

Activation of otherwise unreactive substrates is a common strategy in chemistry and in nature. Adenylate-forming enzymes use adenosine monophosphate to activate the hydroxyl of their carboxylic substrate, creating a better leaving group. In a second step this reactive group is replaced in a nucleophilic elimination reaction to form esters, amides or thioesters. Recent studies have revealed that NRPS- independent siderophore (NIS) synthetases are also adenylate-forming enzymes, but are not included in the current superfamily description. NIS enzymes are involved in biosynthesis of high-affinity iron chelators which are used for iron acquisition by many pathogenic microorganisms. This is an important area of study, not only for potential therapeutic intervention, but also to illuminate new enzyme chemistries. Here the structural and biochemical studies of AcsD from Pectobacterium chrysanthemi are reported. AcsD is a NIS synthetase involved in achromobactin biosynthesis. The co-complex structures of ATP and citrate provide a mechanism for the stereospecific formation of an enzyme-bound citryl-adenylate. This intermediate reacts with L-serine to form a likely achromobactin precursor. A detailed characterization of AcsD nucleophile profile showed that it can not only catalyze ester formation, but also amide and possibly thioester formation, creating new stereospecific citric acid derivatives. The structure of a N-citryl-ethylenediamine product co-complex identifies the residues that are important for both recognition of L-serine and for catalyzing ester formation. The structural studies on the processive enzyme AlcC, which is involved in the final step of alcaligin biosynthesis of Bordetella pertussis, show that it has a similar topology to AcsD. It also shows that ATP is coordinated in a manner similar to that seen in AcsD. Biochemical studies of a substrate analogue establish that AlcC is not only capable of synthesizing substrate dimers and trimers, but also able to assemble the respective dimer and trimer macrocycles. A series of docked binding models have been developed to illustrate the likely substrate coordination and the steps along dimerization and macrocyclization formation. Structural and mechanistic comparison of NIS enzymes with other adenylate-forming enzymes highlights the diversity of the fold, active site architecture, and metal coordination that has evolved. Hence, a new classification scheme for adenylate forming enzymes is proposed.