Conformational switch triggered by α-ketoglutarate in a halogenase of curacin A biosynthesis

Abstract

The CurA halogenase (Hal) catalyzes a cryptic chlorination leading to cyclopropane ring formation in the synthesis of the natural product curacin A. Hal belongs to a family of enzymes that use Fe 2þ , O 2 and α-ketoglutarate (αKG) to perform a variety of halogenation reactions in natural product biosynthesis. Crystal structures of the enzyme in five ligand states reveal strikingly different open and closed conformations dependent on αKG binding. The open form represents ligand-free enzyme, preventing substrate from entering the active site until both αKG and chloride are bound, while the closed form represents the holoenzyme with αKG and chloride coordinated to iron. Candidate amino acid residues involved in substrate recognition were identified by site-directed mutagenesis. These new structures provide direct evidence of a conformational switch driven by αKG leading to chlorination of an early pathway intermediate.cryptic chlorination | natural products | polyketide synthases

Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily

“…Despite conservation of fold, the oligomerization state varies, with AviO1 and EvdO2 monomers, EvdO1 a dimer, and HygX a tetramer. Structural similarity searches support our sequence analysis suggesting that the orthosomycin-associated oxygenases are related to the PhyH subfamily of AKG/Fe(II)-dependent oxygenases (16)(17)(18)(19). Of note, the halogenases SyrB2 and CytC3 cluster near the orthosomycin-associated oxygenases (13,21).…”

“…S1). This orthosomycin-associated subfamily is most closely related to the phytanoyl-CoA 2-hydroxylase (PhyH) subfamily of AKG/Fe(II) oxygenases (16)(17)(18)(19). Our analysis further separates the orthosomycin-associated oxygenases into subgroups (Fig.…”

Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site

“…Examples are the EctD-related structures of the human phytanoyl-CoA hydroxylases PhyH (PDB code 2A1X; r.m.s.d. of 2.6 Å over 252 C-␣ atoms when compared with the SaEctD structure) (55) (59). This structural comparison shows that the core of the EctD protein, its cupin-type fold, is very similar to these proteins; however, the rest of EctD differs from the structurally compared proteins and thereby yields higher r.m.s.d.…”

“…The ectoine hydroxylase (EctD) belongs to this superfamily and mediates the stereo-specific hydroxylation of the compatible solute ectoine to form 5-hydroxyectoine (27,68), which thereby gains novel stress-protective and function-preserving properties (11-15, 35, 36). The crystal structures of EctD from the extremophilic bacteria V. salexigens (44) and S. alaskensis (this study) are most closely related to the human hydroxylases PhyH and PHYD1A (55,56) and to the microbial halogenases SyrB2, CytC3, and CurA (57)(58)(59). It is noteworthy that the substrates of the mentioned halogenases are all tethered via a thioester to the phosphopantetheine arms of acyl carrier proteins of polyketide synthases and nonribosomal peptide synthetases that function within assembly lines directing the synthesis of antibiotics and phytotoxins (57)(58)(59).…”

Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB

“…Significant ligand-dependent protein conformational changes have been observed in several Fe(II)/ 2OG dioxygenases, including AlkB (32), as well as in some homologous halogenases for which crystal structures have been solved in open or closed conformations depending on 2OG binding (33,34). Changes in resonance frequencies and line shapes in 1 H one-dimensional and 1 H-15 N two-dimensional NMR spectra also suggest that the conformational dynamics of AlkB are ligand-dependent (32,35), and these data have been interpreted to reflect increased backbone dynamics that serve to accelerate ligand release from the enzyme upon oxidation of 2OG to Suc.…”