Gilbert, N. C. | Search bioRxiv

bioRxiv2024

The structure of the SufS-SufE complex reveals interactions driving protected persulfide transfer in iron-sulfur cluster biogenesis

Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, PLP-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using E. coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated [~]7{degrees} closer to the EcSufS active site. EcSufE makes clear interactions with the 16 helix of EcSufS and site-directed mutants of several 16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces.

biochemistry↗

bioRxiv2022

Helical remodeling augments 5-lipoxygenase activity

The synthesis of pro-inflammatory leukotrienes implicated in asthma, allergic rhinitis, and atherosclerosis is initiated by the enzyme 5-lipoxygenase (5-LOX). The crystal structure of human Stable-5-LOX revealed a conformation where the catalytic iron was inaccessible to bulk solvent as two aromatic residues on a conserved helix-2 (H2) plugged the substrate access portal. Here, we present a new conformation of 5-LOX where H2 adopts an elongated conformation equivalent to that described in other animal lipoxygenase structures. The sigmoidal kinetic behavior of 5-LOX, which is indicative of positive cooperativity, is consistent with a substrate-induced conformational change that shifts the ensemble of enzyme populations to favor the catalytically competent state. Strategic point mutations along H2 designed to unlock the closed conformation and elongate H2 resulted in improved kinetic parameters, altered limited-proteolysis data, and a drastic reduction in the length of the lag phase yielding the most active 5-LOX enzyme to date. Structural predictions by AlphaFold2 of these variants statistically favor an elongated H2 and reinforce a model in which improved kinetic parameters correlate with a more readily adopted, open conformation.

biochemistry↗