Biochemistry

In bacteria, the ring-shaped sliding clamp, DnaN, is an essential component of the replication machinery. The clamp encircles the parental DNA strand during replication and binds DNA polymerases and other replication and repair proteins, helping to tether them at their site of action on the DNA strand. These binding partners interact with the clamp via short pentapeptide or hexapeptide sequences known as clamp-binding motifs (CBMs). Although conserved CBM sequences have been identified across different bacterial species, most studies of clamp binding have been performed in the model gram-negative bacterium Escherichia coli and less is known about clamp binding in other bacterial species. In this study, we investigate clamp binding in the model gram-positive bacterium Bacillus subtilis. We use fluorescence polarization binding assays to quantify binding of a range of CBM peptides to the clamps of both E. coli and B. subtilis. We identify similarities in clamp binding between the two species, including similar importance of different amino acids within the conserved pentapeptide motif. However, our results also reveal differences in clamp binding between the two species. Most notably, we find that, although pentapeptide CBMs bind the E. coli and B. subtilis clamps with similar affinity, hexapeptide CBMs bind an order of magnitude more weakly to the B. subtilis clamp. Our results provide new insight into clamp binding in bacteria and point to possible species-specific differences in this essential interaction.

The glutaminase (GLS) isoforms KGA and GAC are expressed in neurons where they hydrolyze glutamine to produce the excitatory neurotransmitter glutamate. Two de novo gain-of-function mutants of GLS, S482C and H461L, were recently identified in patients with developmental delay, epilepsy, and infantile cataract. These patients exhibited high glutamate and low glutamine concentrations in the brain, suggesting that the GLS mutants have abnormal enzymology. Here, we examined the enzymatic properties of these GLS mutants and found that they exhibit a total (S482C) or partial (H461L) loss of glutamate product inhibition, lifting this restriction on glutamate accumulation. The mutant enzymes also no longer require the anionic activator phosphate to stimulate enzymatic activity or induce filament formation. Structural analysis of the S482C GAC mutant shows the mutation shifts the key catalytic residue Y466 into the catalytically competent position and disrupts a key hydrogen bond between it and the glutamate product, explaining how the S482C mutant has enzymatic activity in the absence of phosphate and is insensitive to glutamate product inhibition. These results shed new light on the mechanism of phosphate activation and glutamate product inhibition of GLS and show that loss of these enzymatic properties disrupts glutamate homeostasis in the brain and causes neurological disease.

When bacterial ribosomes are assembled in vitro, manipulation of incubation temperature and magnesium ion concentration have been an essential procedure, which is a crucial step for the assembly of active large subunits. The present study tackles with this issue to develop a single-step procedure, which can be performed in a physiological condition. We found that GTPase factors EngA and ObgE can complement the changes in temperature and magnesium ion concentrations. In the presence of these factors, ribosome assembly can proceed under physiological conditions, with magnesium ion concentrations below 10 mM, potassium concentrations around 100 mM, and temperatures of 37 {degrees}C. Both the ribosome assembly and translation processes were successfully integrated in the reconstituted cell-free protein synthesis system. Furthermore, we found that these GTPase factors can reassemble the ribosomes to an active state, whose structure was disrupted by EDTA chelation of magnesium ions, indicating that these two factors can reversibly induce the ribosome structure to an intact state. The findings are essential for the bottom-up construction of synthetic cells.

As an ATP-dependent protease, the quality control functions of Lon have been extensively studied and reviewed in the literature. By contrast, very little research has been conducted to investigate Lons physiological functions and its mechanism as a regulatory protease. In this manuscript, we provided a survey of literature and data to convey that the lambda N ({lambda}N) protein is a suitable Escherichia coli Lon (ELon) substrate for studying the role played by Lon in regulating an RNA transcription process. For proof of principle, we demonstrated that the minimal component of the RNA transcription complex containing RNA polymerase (RNAP) and the {sigma} factor can inhibit {lambda}N degradation by ELon through SDS-PAGE, and the carboxyl-terminal of {lambda}N is important for Lon competing with RNAP interaction. Using negative stain electron microscopy, we obtained structural evidence to show that {lambda}N lacking the carboxyl-terminal flanked by residues 99-107 interacted with ELon differently than full-length {lambda}N. Taken together, the activity and EM data provide a starting point for performing a physiological enzymology study on the contribution of ELon toward RNA transcription.

Adenylate kinases (AKs) are phosphotransferases that are frequently employed as models to investigate protein structure-function relationships. Prior studies have shown that AK homologs of different stabilities retain cellular activity in cells following circular permutation that split the AMP binding domain into fragments coded at different ends of the primary structure, such that this domain was no longer embedded as a continuous polypeptide within the core domain. Herein, we show mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologs at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30{degrees}C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than native AKs, and they exhibited multi-phasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK, and mass spectrometry suggested that the new termini within the AMP binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/564053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ee9ecdorg.highwire.dtl.DTLVardef@fbb415org.highwire.dtl.DTLVardef@ebdd90org.highwire.dtl.DTLVardef@11f4271_HPS_FORMAT_FIGEXP M_FIG C_FIG

Under conditions of oxidative stress or iron starvation, iron-sulfur cluster biogenesis in E. coli is initiated by the cysteine desulfurase, SufS, via the SUF pathway. SufS is a type II cysteine desulfurase that catalyzes the PLP-dependent breakage of an L-cysteine C-S bond to generate L-alanine and a covalent active site persulfide as products. The persulfide is transferred from SufS to SufE and then to the SufBC2D complex, which utilizes it in iron-sulfur cluster biogenesis. Several lines of evidence suggest two conserved arginine residues that line the solvent side of the SufS active site could be important for function. To investigate the mechanistic roles of R56 and R359, the residues were substituted using site-directed mutagenesis to obtain R56A/K and R359A/K SufS variants. Steady state kinetics indicated R56 and R359 have moderate defects in the desulfurase half reaction but major defects in the transpersulfurase step. Fluorescence polarization binding assays showed that the loss of activity was not due to a defect in forming the SufS/SufE complex. Structural characterization of R56A SufS shows loss of electron density for the 3-4 loop at the R56/G57 positions, consistent with a requirement of R56 for proper loop conformation. The structure of R359A SufS exhibits a conformational change in the 3-4 loop allowing R56 to enter the active site and mimics the residues position in the PLP-cysteine aldimine structure. Taken together, the kinetic, binding, and structural data support a mechanism where R359 plays a role in linking SufS catalysis with modulation of the 3-4 loop to promote a close-approach interaction of SufS and SufE conducive to persulfide transfer.

Acetyl-coenzyme A (CoA) is a central metabolic intermediate that links carbon and energy metabolism across all domains of life. The conversion of acetate and acetyl-CoA is carried out by three enzyme pathways: acetate kinase/phosphotransacetylase, ADP-forming acetyl-CoA synthetase, and AMP-forming acetyl-CoA synthetase (Acs). Acs enzymes serve critical physiological roles across diverse organisms generally by catalyzing a reversible two-step reaction forming acetyl-CoA and AMP from acetate and ATP. Isolated from the wastewater reclamation facility in Norman, Oklahoma, Syntrophus aciditrophicus strain SB (Sa) relies on an AMP-forming acetyl-CoA synthetase (SaAcs1) that favors synthesizing acetate and ATP from acetyl-CoA and AMP, in contrast to all previously characterized Acs enzymes. The origin of this preference and the structural determinants of both the thioester-forming step and catalytic directionality remain poorly understood. Here, we report a 2.2 [A] crystal structure of full-length SaAcs1 in the adenylation conformation with acetyl-AMP bound in the active site. Structural comparison to the extensively characterized Acs enzymes from Salmonella enterica (SeAcs) and Cryptococcus neoformans (CnAcs) revealed a displaced CoA-binding loop in SaAcs1. Enzymatic assays confirmed that SaAcs1 preferentially catalyzes the ATP-forming reaction. Site-directed mutagenesis demonstrated that reversion of two residues, G196 and T197, at the beginning of the CoA-binding loop to the consensus sequence repositions the loop and shifts catalytic preference toward the AMP-forming direction. Together, these results establish the CoA-binding loop and G196 and T197 as the primary structural determinants of directional preference in SaAcs1.

Toxin-antitoxin (TA) systems, including YoeB-YefM, are important mediators of bacterial physiological changes. Agrobacterium tumefaciens YoeB and YefM are similar to that from E. coli, and interact as a tight heterotetramer with a KD of 653 pM. We have verified that AtYoeB can perform both ribosome-dependent and -independent RNA cleavage. We have also characterized a newly described metal-dependent and pH-sensitive DNA cleaving ability. We note that this DNA cleaving ability is observed at toxin concentrations as low as 150 nM. The dose-dependence of in vitro ribosome-independent RNA and metal-dependent DNA cleavage is equivalent, and requires a ten-fold increase in toxin concentration as opposed to in the presence of the ribosome. The toxin concentration inside bacterial cells is unknown and according to current models, should increase upon activation of YoeB through degradation of the YefM antitoxin. The discovery of general nuclease activity by AtYoeB, and perhaps other YoeB toxins, offers an opportunity to explore the plasticity of this protein fold and its potential role in the evolution of nucleases.

The surface of gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes are cysteine transpeptidases that recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., Staphylococcus aureus, Streptococcus pyogenes, etc.). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of Staphylococcus aureus sortase A (saSrtA) are the most widely used enzymes. One of the mutated amino acids in the previously engineered pentamutant (or saSrtA5M) enzyme is P94. Structural analyses of experimental saSrtA structures revealed that P94 interacts directly with Y187 when saSrtA is in its inactive conformation. While saSrtA5M, developed via directed evolution, contains a P94R mutation, we wanted to interrogate this position further and ask if other single P94 mutations may reveal a greater effect on activity and/or substrate specificity. We created 18 P94X mutations (excluding P94C), and tested relative activity using a fluorescence resonance energy transfer (FRET) assay for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that outperformed the single mutant P94R for all peptides tested, including P94A, P94D, P94E, P94G, P94H, P94N, P94Q, P94S, and P94T. We further observed that the reactivity of substrates with variations in the central position of the pentapeptide recognition motif (LPXTG) can be sensitive to the identity of the P94X residue. We tested P94A and P94D saSrtA5M variants and found that, depending on LPXTG sequence, these variants could outperform saSrtA5M in activity > 3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw [~]2-fold greater product formation. Taken together, we characterized an important position that modulates substrate access and activity in saSrtA. Furthermore, we argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.

Gram-negative bacteria are intrinsically resistant to many antibiotics in part due to the asymmetric architecture and barrier function of their outer membrane (OM). To establish proper lipid asymmetry, cells need to ensure an intricate balance of constituent OM components, especially lipids. In this regard, the conserved, trans-envelope Tol-Pal complex plays a primary role in maintaining OM lipid homeostasis, thus OM integrity, possibly via retrograde phospholipid transport. However, mechanistic details for this process are unknown, owing to the lack of evidence for direct lipid binding. In this study, we discover that the periplasmic protein TolB, a key component of the Tol-Pal system, associates directly with membranes in vitro, via specific interactions with cardiolipin (CL). Using coarse-grained molecular dynamics simulations, we identify a CL-binding site on TolB; a single amino acid mutation at this site abolishes in vitro membrane interaction, consequently impairing cellular Tol-Pal function in maintaining OM homeostasis in Escherichia coli. Curiously, we find that the functional requirement for TolB-CL interactions can be partially bypassed in cells lacking CL, suggesting compensatory effects through other lipids only when CL is absent. Our findings reveal a previously unappreciated lipid-binding role for TolB, and provide novel insights into how the Tol-Pal complex may facilitate phospholipid transport across the cell envelope. Our work will inform future strategies towards developing new antibiotics against Gram-negative bacteria.

Guanosine 5-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5-monophosphate (XMP) to GMP are comprised of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase and the ammonia thus generated is tunnelled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia tunnelling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channelling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure and cross-linking mass spectrometry guided integrative modelling. pH dependence of enzyme kinetics establish that ammonia is tunnelled across the subunits with the lifetime of the complex being [≤] 0.5 s. The crystal structure of XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/481963v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1eb7261org.highwire.dtl.DTLVardef@a25d02org.highwire.dtl.DTLVardef@1885ed7org.highwire.dtl.DTLVardef@ab189_HPS_FORMAT_FIGEXP M_FIG C_FIG

Computational protein design is growing in popularity as a means to engineer enzymes. Currently, protein design algorithms can predict the stability and function of the enzymes to only a limited degree. Thus, further experimental data is required for training software to more accurately characterize the structure-function relationship of enzymes. To date, the Design2Data (D2D) database holds 129 single point mutations of {beta}-glucosidase B (BglB) characterized by kinetic and thermal stability biophysical parameters. In this study, we introduced six mutants into the BglB database and examined their catalytic activity and thermal stability: L171M, H178M, M221L, E406W, N160E, and F415M.

This study explores computational design predictions related to experimental enzyme behavior by analyzing seven single-point mutants of {beta}-glucosidase B (BglB) from Paenibacillus polymyxa: Y333F, A88E, L219Q, A408H, Y173L, E340S, and Y422F. Each mutation was modeled using Foldit Standalone, and mutant selections were based on predicted thermodynamic stability changes of interest. Six of the seven mutants in this set yielded soluble, expressed protein. Most variants had similar catalytic efficiency compared to the wild type with one exception. The melting temperatures for most variants were also similar to the wild type. Correlation analysis revealed weak but potentially informative relationships between predicted {Delta}TSE and (a) thermal stability and (b) catalytic efficiency. These results further support known limitations of TSE score as a tool for single point mutation design and add to a growing dataset being generated to build the next generation of functionally predictive protein models.

A key goal of protein engineering is to accurately model the stability and catalytic activity of enzymes. However, the limitations of functional predictive abilities pose a major challenge for modeling algorithm design, and can be attributed to the lack of large data sets quantifying the functional properties of enzymes. Here, the thermal stability (TM) and Michaelis-Menten constants (kcat, KM, and kcat/KM) of six new variants of the {beta}-glucosidase B (BglB) protein are quantitatively characterized. Molecular stability of the enzyme variants were hypothesized using the Foldit software and BglB was synthesized in E. coli cells. Testing was done through a colorimetric kinetic assay and thermal stability fluorescence-based protein unfolding assay. Results from the assays suggest that all mutations, with the exception of variant Y169M, all experienced reduced catalytic efficiency compared to the wildtype. Assay results indicate that variant W123R is more thermally stable compared to the wildtype, while the differences in thermal stability between the other variants, and the wildtype are negligible. The collected thermal stability and catalytic efficiency data has been added to a data set with the aim of improving Rosetta algorithms for modeling and predicting the functional interactions between biomolecules through a machine learning algorithm and facilitate the precise engineering of protein catalysts.

Multi-functionality in extant enzymes, including the ability to transform multiple substrates, is thought to arise, in part, from conformational flexibility. The hexokinase protein family represents a classic model system for investigating the relationship between substrate specificity and conformational change. Within this family, human glucokinase (hGCK) displays notable degrees of conformational heterogeneity, including an intrinsically disordered loop. The extent to which these structural features contribute to the breadth of hGCKs substrate scope is unknown. Here, we investigate the substrate specificities of extant and ancestral glucokinases that span the evolutionary emergence of conformational heterogeneity in this family. We show that extant hGCK catalyzes the ATP-dependent phosphorylation of glucose, 2-deoxyglucose, mannose, glucosamine, fructose, allose and galactose with catalytic efficiencies ranging from 6.3 x 103 M-1 sec-1 to 0.33 M-1sec-1. A glucokinase ancestor from early vertebrate evolution (vGCK), which also displays conformational heterogeneity and disorder, phosphorylates these same seven substrates with similar kcat/Km values. An antecedent, chordate glucokinase (cGCK), which displays reduced conformational heterogeneity and lacks intrinsic disorder, also transforms these same substrates, but with higher overall catalytic efficiencies and markedly lower Km values. Notably, however, the ratios of kcat/Km values for individual substrate pairs, which define specificity, are unchanged for all three enzymes. Our results demonstrate that substrate specificity is not correlated with conformational diversity in GCKs and support a model in which the differences in catalytic efficiencies of various substrates arise from differences in the ability to form the ground state enzyme-carbohydrate binary complex.

Antimicrobial resistant infections present a growing threat to public health and were associated with or directly caused 6 million deaths globally in 2021. This huge death toll highlights the need for novel strategies to address AMR infections. Interfering with the regulation of resistance mechanisms could provide an alternative approach to treat drug-resistant infections. PhoQ, a sensor histidine kinase ubiquitous amongst gram-negative bacteria, regulates several virulence factors, as well as resistance to outer membrane-targeting antibiotics, making it an attractive target for adjuvant therapy development. However, the identification of potent small molecule inhibitors is limited by the assays available for in vitro assessment of binding and activity inhibition in PhoQ. Thus, we sought to investigate the use of a fluorescence-based assay to evaluate enzymatic activity, as well as a thermal shift assay to assess inhibitor-protein binding in PhoQ. Together, these newly implemented protocols are valuable contributors to the toolbox of methods available for the development of PhoQ-targeted inhibitors to block this major contributor to antimicrobial resistance.

Overexpression of the multidrug efflux pump MtrCDE, a critical factor of multidrug-resistance in Neisseria gonorrhoeae, the causative agent of gonorrheae, is repressed by the transcriptional regulator, MtrR (multiple transferable resistance repressor). Here, we report the results from a series of in vitro experiments to identify innate, human inducers of MtrR and to understand the biochemical and structural mechanisms of the gene regulatory function of MtrR. Isothermal titration calorimetry experiments reveal that MtrR binds the hormonal steroids progesterone, {beta}-estradiol, and testosterone, all of which are present at significant concentrations at urogenital infection sites as well as ethinyl estrogen, a component of some birth control pills. Binding of these steroids results in decreased affinity of MtrR for cognate DNA, as demonstrated by fluorescence polarization-based assays. The crystal structures of MtrR bound to each steroid provided insight into the flexibility of the binding pocket, elucidated specific residue-ligand interactions, and revealed the conformational consequences of the induction mechanism of MtrR. Three residues, D171, W136 and R176 are key to the specific binding of these gonadal steroids. These studies provide a molecular understanding of the transcriptional regulation by MtrR that promotes N. gonorrhoeae survival in its human host.

Though ribosomes have several features that help them maintain their reading frame, these safeguards can be bypassed by RNA structures that promote -1 programmed ribosomal frameshifting (-1PRF). We recently found that conformational transitions in the nascent polypeptide can enhance -1PRF, though its unclear whether this feedback plays a general role in translational recoding. Here we demonstrate that the translocation of nascent transmembrane domains is sufficient to induce -1PRF during the decoding of slippery heptamers. We identify thousands of motifs that potentially trigger -1PRF along with proteomic identifications of 33 predicted human frameshift products. We also identify thousands of splicing-dependent motifs and demonstrate that the splicing-mediated reconfiguration of transmembrane domains alters -1PRF. Finally, we show that most transcripts bearing these motifs are sensitive to the nonsense-mediated decay regulator UPF1, suggesting they modulate mRNA turnover. Our findings show that the misassembly of growing polypeptides can trigger -1PRF, premature termination, and transcript decay.

S-acylation is the addition of fatty acids to cysteine residues to regulate protein function and localization. S-acylation is catalyzed by the ZDHHC (Asp-His-His-Cys) family of protein S-acyltransferases (PATs), which S-acylate protein substrates by first auto-S-acylating the catalytic cysteine of the DHHC active site followed by transfer to the substrate. ZDHHC13 and ZDHHC17 are related ankyrin repeat domain (ANK) PATs that S-acylate multiple neuronal proteins, including huntingtin (HTT), the protein mutated in Huntington disease. However, unlike ZDHHC17 and other human PATs, ZDHHC13 possesses a non-canonical DQHC active site. As the first histidine is essential for auto-S-acylation, it is unclear if ZDHHC13 is catalytically active. Our phylogenetic analysis of eukaryotic ANK-containing PATs shows that ZDHHC13 orthologues are more divergent compared to ZDHHC17. While the ZDHHC17 DHHC is highly conserved, the motif varies among ZDHHC13 orthologues, with some vertebrate lineages containing a serine in place of the catalytic cysteine. Interestingly, we found that the ZDHHC13 S-acylation is lower than that of ZDHHC17, but the ZDHHC13 catalytic cysteine is indeed S-acylated. While expression of wild type (WT) ZDHHC13 in ZDHHC13 deficient HEK293T cells increased S-acylation of a HTT1-588 fragment, surprisingly, expression of catalytically dead DQHS ZDHHC13 was still able to facilitate HTT1-588 S-acylation equally. This suggests the ZDHHC13 catalytic cysteine is not required for S-acylation of target proteins, suggesting ZDHHC13 may coordinate another PAT. Indeed, we identified ZDHHC13 in high-molecular weight complexes. Our results indicate that ZDHHC13 is a likely pseudoenzyme that may function via a non-conventional mechanism reliant on other PATs. This work broadens our understanding of the function of this non-canonical PAT.

Polyketide synthases (PKSs) are megaenzymes that form chemically diverse polyketides and are found within the genomes of nearly all classes of life. We recently discovered the type I PKS from the apicomplexan parasite Toxoplasma gondii, TgPKS2, which contains a unique putative chain release mechanism that includes ketosynthase (KS) and thioester reductase (TR) domains. Our bioinformatic analysis of the thioester reductase of TgPKS2, TgTR, suggests differences in putative apicomplexan reductase domains compared to other systems and hints at a possibly conserved release mechanism within the apicomplexan subclass Coccidia. To evaluate this release module, we first isolated TgTR and observed that it is capable of 4 electron (4e-) reduction of octanoyl-CoA to the primary alcohol, octanol, utilizing NADH as a cofactor. TgTR was also capable of generating octanol in the presence of octanal and NADH, but no reactions were observed when NADPH was supplied as a cofactor. To biochemically characterize the protein, we measured the catalytic efficiency of TgTR using a fluorescence assay and determined the TgTR binding affinity for cofactor and substrates using isothermal titration calorimetry (ITC). We additionally show that TgTR is capable of reducing an acyl carrier protein (ACP)-tethered substrate by liquid chromatography mass spectrometry and determine that TgTR binds to holo-TgACP4, its predicted cognate ACP, with a KD of 5.75 {+/-} 0.77 {micro}M. Finally, our transcriptional analysis shows that TgPKS2 is upregulated [~]4-fold in the parasites cyst-forming bradyzoite stage compared to tachyzoites. Our study identifies features that distinguish TgPKS2 from well-characterized systems in bacteria and fungi, and suggests it aids the T. gondii cyst stage. Together, this work increases our knowledge of PKS thioester reductase domains and advances our understanding of unconventional polyketide chain termination mechanisms.