Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme.

Many peroxy-containing secondary metabolites have been isolated and shown to provide beneficial effects to human health. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.

Dynamics underlie the drug recognition mechanism by the efflux transporter EmrE.

The multidrug efflux transporter EmrE from Escherichia coli requires anionic residues in the substrate binding pocket for coupling drug transport with the proton motive force. Here, we show how protonation of a single membrane embedded glutamate residue (Glu14) within the homodimer of EmrE modulates the structure and dynamics in an allosteric manner using NMR spectroscopy. The structure of EmrE in the Glu14 protonated state displays a partially occluded conformation that is inaccessible for drug binding by the presence of aromatic residues in the binding pocket. Deprotonation of a single Glu14 residue in one monomer induces an equilibrium shift toward the open state by altering its side chain position and that of a nearby tryptophan residue. This structural change promotes an open conformation that facilitates drug binding through a conformational selection mechanism and increases the binding affinity by approximately 2000-fold. The prevalence of proton-coupled exchange in efflux systems suggests a mechanism that may be shared in other antiporters where acid/base chemistry modulates access of drugs to the substrate binding pocket.

Assessing Interactions Between a Polytopic Membrane Protein and Lipid Bilayers Using Differential Scanning Calorimetry and Solid-State NMR.

It is known that the lipid composition within a cellular membrane can influence membrane protein structure and function. In this Article, we investigated how structural changes to a membrane protein upon substrate binding can impact the lipid bilayer. To carry out this study, we reconstituted the secondary active drug transporter EmrE into a variety of phospholipid bilayers varying in headgroup and chain length and carried out differential scanning calorimetry (DSC) and solid-state NMR experiments. The DSC results revealed a difference in cooperativity of the lipid phase transition for drug-free EmrE protonated at glutamic acid 14 (i.e., proton-loaded form) and the tetraphenylphosphonium (TPP+) bound form of the protein (i.e., drug-loaded form). To complement these findings, we acquired magic-angle-spinning (MAS) spectra in the presence and absence of TPP+ by directly probing the phospholipid headgroup using 31P NMR. These spectra showed a reduction in lipid line widths around the main phase transition for samples where EmrE was bound to TPP+ compared to the drug free form. Finally, we collected oriented solid-state NMR spectra on isotopically enriched EmrE that displayed chemical shift perturbations to both transmembrane and loop residues upon TPP+ binding. All of these results prompt us to propose a mechanism whereby substrate-induced changes to the structural dynamics of EmrE alters the surrounding lipids within the bilayer.

Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate.

Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.

Cysteine oxidation reactions catalyzed by a mononuclear non-heme iron enzyme (OvoA) in ovothiol biosynthesis.

OvoA in ovothiol biosynthesis is a mononuclear non-heme iron enzyme catalyzing the oxidative coupling between histidine and cysteine. It can also catalyze the oxidative coupling between hercynine and cysteine, yet with a different regio-selectivity. Due to the potential application of this reaction for industrial ergothioneine production, in this study, we systematically characterized OvoA by a combination of three different assays. Our studies revealed that OvoA can also catalyze the oxidation of cysteine to either cysteine sulfinic acid or cystine. Remarkably, these OvoA-catalyzed reactions can be systematically modulated by a slight modification of one of its substrates, histidine.