Understanding and improving the oxygen-tolerance of Thi4 metalloenzymes
Andrew D. Hanson, University of Florida (Principal Investigator)
Mark A. Wilson, University of Nebraska-Lincoln (Co-Investigator)
Joseph D. Yesselman University of Nebraska-Lincoln (Collaborator)
Background. Thi4 enzymes synthesize the thiazole moiety of the cofactor thiamin diphosphate, which is central to carbon and energy flows in photosynthesis and respiration. The thiazole moiety is labile and so must be continually resynthesized. All Thi4s mediate a multistep reaction in which the thiazole ring is built from the ribose of NAD+, glycine, and a sulfur atom. Canonical plant Thi4s are suicide Zn(II) enzymes that use an active-site cysteine as sulfur donor and hence self-inactivate after a single reaction cycle. The combination of a suicidal Thi4 and a ceaseless demand for thiazole forces plants to divert as much as 2-4% of their total energy budget to support Thi4 protein turnover.
Many prokaryotes have catalytic (i.e., non-suicidal) Thi4s that can use sulfide as sulfur donor and are potential replacements for the energy-costly suicide Thi4s of plants. However, these Thi4s are Fe(II) enzymes that require low O2 levels and high sulfide for activity and are very slow; hence they require substantial improvement if they are to work in plants. Our experimental and genomic data for catalytic Thi4s with a degree of natural O2-tolerance point to oxidative attack on the Fe(II) center and nearby residues as drivers of inactivation by O2. Directed evolution of such Thi4s shows that single mutations improve performance in air at moderate sulfide levels, and that additional mutations give further improvements. Experimental and genomic data also show – surprisingly – that cereals (unlike other plants) have both a suicide Thi4 and a catalytic one that, like bacterial catalyticThi4s, needs a low O2 level to function. Genome analysis predicts – surprisingly again – that some bacterial Thi4s use an activated form of sulfur as sulfur donor. This could be an unrecognized major path to catalytic operation in the presence of O2 that avoids sulfide toxicity.
Objectives. We now propose to push evolution of prokaryote Thi4s much further, to evolve plant catalytic Thi4s, to mechanistically understand the improvements that emerge, and to find if using an activated form of sulfur (persulfide or thiocarboxylate) as physiological sulfur donor is the rule or an exception. Specifically, we will:
1. Evolve bacterial / plant Thi4s for more O2-tolerance (i.e., less inactivation) and activity in low sulfide
2. Use cryo-EM and X-ray crystallography to determine 3D structures throughout evolution campaigns
3. Combine structure data and molecular dynamics simulations to uncover improvement mechanisms
4. Apply these mechanisms to design new mutational improvements, then test and further evolve them
5. Determine whether bacterial Thi4s prefer or require a persulfide or thiocarboxylate as sulfur donor
Description. We will use the yeast OrthoRep continuous directed evolution system to further increase activity in aerobic conditions of our existing mutant bacterial Thi4 mutants, first at elevated sulfide, then at low sulfide, plus or minus a toxic thiazole analog. We will do likewise with catalytic plant Thi4s and with wildtype Thi4s from bacteria whose ecology and genomes indicate substantial O2-tolerance.
During evolution campaigns we will isolate improved variants from the populations and identify the mutations responsible. Thi4s have very low kcat values and are highly O2-labile in vitro, precluding biochemical characterization. We will therefore take structure- and simulation-based approaches. Structures of variant Thi4s along evolutionary paths will be determined via cryo-EM, supplemented by X-ray crystallography. We expect to identify oxidatively damaged residues and so get direct mechanistic evidence on inactivation by O2 and its mitigation. We will apply molecular dynamics (MD) simulations to our Thi4 structures to further explore interactions with O2, the sulfur donor, and other substrates. We will then extract heuristic rules to predict features affecting inactivation by O2, apply them to design improvements to wildtype Thi4s, and test the O2-tolerance of the designed enzymes.
We will test for a persulfide or thiocarboxylate sulfur donor via surrogate genetics in a thiazole-requiring Escherichia coli strain by encoding different combinations of predicted sulfur transfer chain components (e.g., cysteine desulfurase, sulfur carrier, ThiF, ThiS) and a putative client Thi4 on a plasmid, and testing for growth. Positive findings will be followed up biochemically.
Potential Impact. This research will elucidate the structure and mechanism of Thi4s that have evolved the remarkable ability to act catalytically in O2-rich conditions using sulfide (or its activated form) as sulfur donor. More broadly, it will suggest how to engineer Fe(II) enzymes to resist oxidative damage, and provide knowledge needed to design energy-efficient Thi4s to trial in energy crops.
