Biology subjects

Boal, A. K.

Publications and source records attributed to Boal, A. K..

3 recordsLinked to original sources

bioRxiv2023

Synergistic Binding of the Halide and Cationic Prime Substrate of the L-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl- or Br-), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three non-gaseous substrates must bind to activate the enzymes Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2 all coordinate directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H*) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the O_SCPLOWLC_SCPLOW-lysine 4-chlorinase, BesD. After 2OG adds, subsequent coordination of the halide to the cofactor and binding of cationic O_SCPLOWLC_SCPLOW-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon addition of O2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and O_SCPLOWLC_SCPLOW-Lys. The surprising lability of the BesD*[Fe(IV)=O]*Cl*succinate*O_SCPLOWLC_SCPLOW-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in O_SCPLOWLC_SCPLOW-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply that (i) BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between O_SCPLOWLC_SCPLOW-Lys binding and chloride coordination following loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

biochemistry↗

bioRxiv2021

Structural Basis for an Unprecedented Enzymatic Alkylation in Cylindrocyclophane Biosynthesis

The cyanobacterial enzyme CylK assembles the cylindrocyclophane natural products by performing two unusual alkylation reactions, forming new carbon-carbon bonds between aromatic rings and secondary alkyl halide substrates. This transformation is unprecedented in biology and the structure and mechanism of CylK are unknown. Here, we report x-ray crystal structures of CylK, revealing a distinctive fusion of a Ca2+ binding domain and a {beta}-propeller fold. We use a mutagenic screening approach to locate CylKs active site at its domain interface, identifying two residues, Arg105 and Tyr473, that are required for catalysis. Anomalous diffraction datasets collected with bound bromide ions, a product analog, suggest these residues interact with the alkyl halide electrophile. Additional mutagenesis and molecular dynamics simulations implicates Asp440 and Glu374 in activating the nucleophilic aromatic ring. Bioinformatic analysis of CylK homologs from other cyanobacteria establishes that they conserve these key catalytic amino acids but they are likely associated with divergent reactivity and altered secondary metabolism. By gaining a molecular understanding of this unusual biosynthetic transformation, this work fills a gap in our understanding of how alkyl halides are activated and used by enzymes as biosynthetic intermediates, informing enzyme engineering, catalyst design, and natural product discovery.

biochemistry↗

bioRxiv2020

Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF

In biosynthesis of the pancreatic cancer drug streptozotocin, the tri-domain nonheme-iron oxygenase, SznF, hydroxylates N{delta} and N{omega} of N{omega}-methyl-L-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the mono-iron cofactor in the enzymes C-terminal cupin domain, which effects the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. Here we leverage our recent report of an intensely absorbing {micro}-peroxodiiron(III/III) intermediate formed from the Fe2(II/II) complex and O2 to understand assembly of the diiron cofactor in the HO-like domain and to obtain structures with both SznF iron cofactors bound. Tight binding at one diiron subsite is associated with a conformational change, which is followed by weak binding at the second subsite and rapid capture of O2 by the Fe2(II/II) complex. Differences between iron-deficient and iron-replete structures reveal both the conformational change required to form the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability, showing that a ligand-harboring core helix dynamically refolds during metal acquisition and release. The cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulation. The additional ligand is conserved in another experimentally validated HO-like N-oxygenase but not in two known HO-like diiron desaturases. Among [~]9600 sequences identified bioinformatically as belonging to the emerging HO-like diiron protein (HDO) superfamily, [~]25% have this carboxylate residue and are thus tentatively assigned as N-oxygenases. Significance statementThe enzyme SznF assembles the N-nitrosourea pharmacophore of the drug streptozotocin. Its central N-oxygenase domain resembles heme-oxygenase (HO) and belongs to an emerging superfamily of HO-like diiron enzymes (HDOs) with unstable metallocofactors that have resisted structural characterization. We investigated assembly of the O2-reactive diiron complex from metal-free SznF and Fe(II) and leveraged this insight to obtain the first structure of a functionally assigned HDO with intact cofactor. Conformational changes accompanying cofactor acquisition explain its instability, and the observation of an unanticipated glutamate ligand that is conserved in only a subset of the HDO sequences provides a potential basis for top-level assignment of enzymatic function. Our results thus provide a roadmap for structural and functional characterization of novel HDOs.

biochemistry↗