ABSTRACT
Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the termina PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TE DAP ) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TE DAP , the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 Å from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide new insights to facilitate TE engineering and optimization.
ABSTRACT
G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue and cellular levels. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays and structural studies, we develop maternally selective heavy-chain-only antibody ('nanobody') antagonists against the angiotensin II type I receptor and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to angiotensin II type I receptor with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.
ABSTRACT
Adhesion G protein-coupled receptor (aGPCR) signaling influences development and homeostasis in a wide range of tissues. In the current model for aGPCR signaling, ligand binding liberates a conserved sequence that acts as an intramolecular, tethered agonist (TA), yet this model has not been evaluated systematically for all aGPCRs. Here, we assessed the TA-dependent activities of all 33 aGPCRs in a suite of transcriptional reporter, G protein activation, and ß-arrestin recruitment assays using a new fusion protein platform. Strikingly, only â¼50% of aGPCRs exhibited robust TA-dependent activation, and unlike other GPCR families, aGPCRs showed a notable preference for G12/13 signaling. AlphaFold2 predictions assessing TA engagement in the predicted intramolecular binding pocket aligned with the TA dependence of the cellular responses. This dataset provides a comprehensive resource to inform the investigation of all human aGPCRs and for targeting aGPCRs therapeutically.
Subject(s)
Receptors, G-Protein-Coupled , Signal Transduction , Humans , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/agonists , Signal Transduction/drug effects , HEK293 Cells , LigandsABSTRACT
The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor (GPCR) that regulates important physiologic processes, including vascular tone, bronchoconstriction, and insulin secretion. It is expressed on a wide variety of cell types, including pancreatic beta, smooth muscle, neuronal, and immune cells. Agonist binding to the M3R is thought to initiate intracellular signaling events primarily through the heterotrimeric G protein Gq. However, reports differ on the ability of M3R to couple to other G proteins beyond Gq. Using members from the four primary G protein families (Gq, Gi, Gs, and G13) in radioligand binding, GTP turnover experiments, and cellular signaling assays, including live cell G protein dissociation and second messenger assessment of cAMP and inositol trisphosphate, we show that other G protein families, particularly Gi and Gs, can also interact with the human M3R. We further show that these interactions are productive as assessed by amplification of classic second messenger signaling events. Our findings demonstrate that the M3R is more promiscuous with respect to G protein interactions than previously appreciated. SIGNIFICANCE STATEMENT: The study reveals that the human M3 muscarinic acetylcholine receptor (M3R), known for its pivotal roles in diverse physiological processes, not only activates intracellular signaling via Gq as previously known but also functionally interacts with other G protein families such as Gi and Gs, expanding our understanding of its versatility in mediating cellular responses. These findings signify a broader and more complex regulatory network governed by M3R and have implications for therapeutic targeting.
Subject(s)
GTP-Binding Proteins , Receptor, Muscarinic M3 , Signal Transduction , Animals , Humans , CHO Cells , Cricetulus , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , GTP-Binding Proteins/metabolism , HEK293 Cells , Receptor, Muscarinic M3/metabolism , Signal Transduction/physiologyABSTRACT
G protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue, and cellular level. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays, and structural studies, we develop maternally selective heavy chain-only antibody ("nanobody") antagonists against the angiotensin II type I receptor (AT1R) and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to AT1R with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.
ABSTRACT
Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. Here, we design a set of experiments using a diverse naïve synthetic camelid antibody fragment (nanobody) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally test our models' performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the models allow us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its functional properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.
Subject(s)
Immunoglobulin FragmentsABSTRACT
Construction and remodeling of the bacterial peptidoglycan (PG) cell wall must be carefully coordinated with cell growth and division. Central to cell wall construction are hydrolases that cleave bonds in peptidoglycan. These enzymes also represent potential new antibiotic targets. One such hydrolase, the amidase LytH in Staphylococcus aureus, acts to remove stem peptides from PG, controlling where substrates are available for insertion of new PG strands and consequently regulating cell size. When it is absent, cells grow excessively large and have division defects. For activity, LytH requires a protein partner, ActH, that consists of an intracellular domain, a large rhomboid protease domain, and three extracellular tetratricopeptide repeats (TPRs). Here, we demonstrate that the amidase-activating function of ActH is entirely contained in its extracellular TPRs. We show that ActH binding stabilizes metals in the LytH active site and that LytH metal binding in turn is needed for stable complexation with ActH. We further present a structure of a complex of the extracellular domains of LytH and ActH. Our findings suggest that metal cofactor stabilization is a general strategy used by amidase activators and that ActH houses multiple functions within a single protein.
Subject(s)
Bacterial Proteins , Membrane Proteins , Metals , N-Acetylmuramoyl-L-alanine Amidase , Bacterial Proteins/chemistry , Cell Wall/chemistry , Enzyme Activation , Enzyme Stability , Membrane Proteins/chemistry , Metals/chemistry , N-Acetylmuramoyl-L-alanine Amidase/chemistry , Peptidoglycan/chemistry , Protein Binding , Protein DomainsABSTRACT
Installation of methyl groups can significantly improve the binding of small-molecule drugs to protein targets; however, site-selective methylation often presents a significant synthetic challenge. Metal- and S-adenosyl-methionine (SAM)-dependent methyltransferases (MTs) in natural-product biosynthetic pathways are powerful enzymatic tools for selective or chemically challenging C-methylation reactions. Each of these MTs selectively catalyzes one or two methyl transfer reactions. Crystal structures and biochemical assays of the Mn2+-dependent monomethyltransferase from the saxitoxin biosynthetic pathway (SxtA MT) revealed the structural basis for control of methylation extent. The SxtA monomethyltransferase was converted to a dimethyltransferase by modification of the metal binding site, addition of an active site base, and an amino acid substitution to provide space in the substrate pocket for two methyl substituents. A reciprocal change converted a related dimethyltransferase into a monomethyltransferase, supporting our hypothesis that steric hindrance can prevent a second methylation event. A novel understanding of MTs will accelerate the development of MT-based catalysts and MT engineering for use in small-molecule synthesis.
Subject(s)
Methyltransferases , Polyketide Synthases , Catalytic Domain , Methylation , Methyltransferases/metabolism , Polyketide Synthases/metabolism , Protein Domains , S-Adenosylmethionine/metabolismABSTRACT
Endogenous self-reactive autoantibodies (AAs) recognize a range of G-protein-coupled receptors (GPCRs). They are frequently associated with cardiovascular, neurological, and autoimmune disorders, and in some cases directly impact disease progression. Many GPCR AAs modulate receptor signaling, but molecular details of their modulatory activity are not well understood. Technological advances have provided insight into GPCR biology, which now facilitates deeper understanding of GPCR AA function at the molecular level. Most GPCR AAs are allosteric modulators and exhibit a broad range of pharmacological properties, altering both receptor signaling and trafficking. Understanding GPCR AAs is not only important for defining how these unusual GPCR modulators function in disease, but also provides insight into the potential use and limitations of using therapeutic antibodies to modulate GPCR signaling.
Subject(s)
Autoantibodies , Autoimmune Diseases , Allosteric Regulation , Autoimmune Diseases/drug therapy , Humans , Receptors, G-Protein-CoupledABSTRACT
The σ1 receptor is a transmembrane protein implicated in several pathophysiological conditions, including neurodegenerative disease (J. Pharmacol. Sci.2015127 (1), 1729), drug addiction (Behav. Pharmacol.201627 (2-3 Spec Issue), 10015), cancer (Handb. Exp. Pharmacol.2017244237308), and pain (Neural Regener. Res.201813 (5), 775778). However, there are no high-throughput functional assays for σ1 receptor drug discovery. Here, we assessed high-throughput structure-based computational docking for discovery of novel ligands of the σ1 receptor. We screened a library of over 6 million compounds using the Schrödinger Glide package, followed by experimental characterization of top-scoring candidates. 77% of tested candidates bound σ1 with high affinity (KD < 1 µM). These include compounds with high selectivity for the σ1 receptor compared to the genetically unrelated but pharmacologically similar σ2 receptor, as well as compounds with substantial crossreactivity between the two receptors. These results establish structure-based virtual screening as a highly effective platform for σ1 receptor ligand discovery and provide compounds to prioritize in studies of σ1 biology.
ABSTRACT
There is considerable interest in developing antibodies as functional modulators of G protein-coupled receptor (GPCR) signaling for both therapeutic and research applications. However, there are few antibody ligands targeting GPCRs outside of the chemokine receptor group. GPCRs are challenging targets for conventional antibody discovery methods, as many are highly conserved across species, are biochemically unstable upon purification, and possess deeply buried ligand-binding sites. Here, we describe a selection methodology to enrich for functionally modulatory antibodies using a yeast-displayed library of synthetic camelid antibody fragments called "nanobodies." Using this platform, we discovered multiple nanobodies that act as antagonists of the angiotensin II type 1 receptor (AT1R). Following angiotensin II infusion in mice, we found that an affinity matured nanobody antagonist has comparable antihypertensive activity to the angiotensin receptor blocker (ARB) losartan. The unique pharmacology and restricted biodistribution of nanobody antagonists may provide a path for treating hypertensive disorders when small-molecule drugs targeting the AT1R are contraindicated, for example, in pregnancy.
Subject(s)
Angiotensin Receptor Antagonists , Receptors, Angiotensin/immunology , Single-Domain Antibodies , Animals , Antibody Affinity , Blood Pressure , Cell Line , Humans , MiceABSTRACT
Biased signaling, in which different ligands that bind to the same G protein-coupled receptor preferentially trigger distinct signaling pathways, holds great promise for the design of safer and more effective drugs. Its structural mechanism remains unclear, however, hampering efforts to design drugs with desired signaling profiles. Here, we use extensive atomic-level molecular dynamics simulations to determine how arrestin bias and G protein bias arise at the angiotensin II type 1 receptor. The receptor adopts two major signaling conformations, one of which couples almost exclusively to arrestin, whereas the other also couples effectively to a G protein. A long-range allosteric network allows ligands in the extracellular binding pocket to favor either of the two intracellular conformations. Guided by this computationally determined mechanism, we designed ligands with desired signaling profiles.
Subject(s)
Arrestins/chemistry , GTP-Binding Proteins/chemistry , Receptor, Angiotensin, Type 1/chemistry , Signal Transduction , Allosteric Regulation , Humans , Molecular Dynamics Simulation , Protein ConformationABSTRACT
Biased agonists of G protein-coupled receptors (GPCRs) preferentially activate a subset of downstream signaling pathways. In this work, we present crystal structures of angiotensin II type 1 receptor (AT1R) (2.7 to 2.9 angstroms) bound to three ligands with divergent bias profiles: the balanced endogenous agonist angiotensin II (AngII) and two strongly ß-arrestin-biased analogs. Compared with other ligands, AngII promotes more-substantial rearrangements not only at the bottom of the ligand-binding pocket but also in a key polar network in the receptor core, which forms a sodium-binding site in most GPCRs. Divergences from the family consensus in this region, which appears to act as a biased signaling switch, may predispose the AT1R and certain other GPCRs (such as chemokine receptors) to adopt conformations that are capable of activating ß-arrestin but not heterotrimeric Gq protein signaling.
Subject(s)
Angiotensin II/chemistry , Receptor, Angiotensin, Type 1/chemistry , Humans , Ligands , Protein Conformation , Signal Transduction , beta-Arrestins/chemistryABSTRACT
Natural product biosynthetic pathways are replete with enzymes repurposed for new catalytic functions. In some modular polyketide synthase (PKS) pathways, a GCN5-related N-acetyltransferase (GNAT)-like enzyme with an additional decarboxylation function initiates biosynthesis. Here, we probe two PKS GNAT-like domains for the dual activities of S-acyl transfer from coenzyme A (CoA) to an acyl carrier protein (ACP) and decarboxylation. The GphF and CurA GNAT-like domains selectively decarboxylate substrates that yield the anticipated pathway starter units. The GphF enzyme lacks detectable acyl transfer activity, and a crystal structure with an isobutyryl-CoA product analog reveals a partially occluded acyltransfer acceptor site. Further analysis indicates that the CurA GNAT-like domain also catalyzes only decarboxylation, and the initial acyl transfer is catalyzed by an unidentified enzyme. Thus, PKS GNAT-like domains are re-classified as GNAT-like decarboxylases. Two other decarboxylases, malonyl-CoA decarboxylase and EryM, reside on distant nodes of the superfamily, illustrating the adaptability of the GNAT fold.
Subject(s)
Acetyltransferases/chemistry , Acetyltransferases/metabolism , Polyketides/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Protein Domains , Protein Folding , Protein Structure, SecondaryABSTRACT
Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate ß-hydroxyl or ß-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite ß-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis.
Subject(s)
Methyltransferases/chemistry , Polyketide Synthases/chemistry , Polyketides/chemical synthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Catalytic Domain/genetics , Cyanobacteria/enzymology , Methylation , Methyltransferases/genetics , Mutagenesis, Site-Directed , Mutation , Myxococcales/enzymology , Polyketide Synthases/genetics , Protein Conformation , Protein Domains , Substrate SpecificityABSTRACT
The structural diversity and complexity of marine natural products have made them a rich and productive source of new bioactive molecules for drug development. The identification of these new compounds has led to extensive study of the protein constituents of the biosynthetic pathways from the producing microbes. Essential processes in the dissection of biosynthesis have been the elucidation of catalytic functions and the determination of 3D structures for enzymes of the polyketide synthases and nonribosomal peptide synthetases that carry out individual reactions. The size and complexity of these proteins present numerous difficulties in the process of going from gene to structure. Here, we review the problems that may be encountered at the various steps of this process and discuss some of the solutions devised in our and other labs for the cloning, production, purification, and structure solution of complex proteins using Escherichia coli as a heterologous host.
Subject(s)
Peptide Synthases/genetics , Polyketide Synthases/genetics , Protein Engineering/methods , Recombinant Proteins/isolation & purification , Acyl Carrier Protein/genetics , Acyl Carrier Protein/metabolism , Bacteria/genetics , Bacterial Outer Membrane Proteins/metabolism , Cloning, Molecular/methods , Codon , Crystallization , Culture Media/chemistry , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli Proteins/metabolism , Peptide Synthases/chemistry , Peptide Synthases/isolation & purification , Peptide Synthases/metabolism , Plasmids/genetics , Polyketide Synthases/chemistry , Polyketide Synthases/isolation & purification , Polyketide Synthases/metabolism , Promoter Regions, Genetic , Protein Domains , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.
Subject(s)
Bacterial Proteins/metabolism , Carboxy-Lyases/metabolism , Depsipeptides/biosynthesis , Methyltransferases/metabolism , Multifunctional Enzymes/metabolism , Polyketide Synthases/metabolism , Acyl Carrier Protein/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Carboxy-Lyases/chemistry , Catalytic Domain , Cyanobacteria/chemistry , Decarboxylation , Depsipeptides/chemistry , Depsipeptides/isolation & purification , Methylation , Methyltransferases/chemistry , Multifunctional Enzymes/chemistry , Polyketide Synthases/chemistry , Substrate SpecificityABSTRACT
Like many complex natural products, the intricate architecture of saxitoxin (STX) has hindered full exploration of this scaffold's utility as a tool for studying voltage-gated sodium ion channels and as a pharmaceutical agent. Established chemical strategies can provide access to the natural product; however, a chemoenzymatic route to saxitoxin that could provide expedited access to related compounds has not been devised. The first step toward realizing a chemoenzymatic approach toward this class of molecules is the elucidation of the saxitoxin biosynthetic pathway. To date, a biochemical link between STX and its putative biosynthetic enzymes has not been demonstrated. Herein, we report the first biochemical characterization of any enzyme involved in STX biosynthesis. Specifically, the chemical functions of a polyketide-like synthase, SxtA, from the cyanobacteria Cylindrospermopsis raciborskii T3 are elucidated. This unique megasynthase is comprised of four domains: methyltransferase (MT), GCN5-related N-acetyltransferase (GNAT), acyl carrier protein (ACP), and the first example of an 8-amino-7-oxononanoate synthase (AONS) associated with a multidomain synthase. We have established that this single polypeptide carries out the formation of two carbon-carbon bonds, two decarboxylation events and a stereospecific protonation to afford the linear biosynthetic precursor to STX (4). The synthetic utility of the SxtA AONS is demonstrated by the synthesis of a suite of α-amino ketones from the corresponding α-amino acid in a single step.
Subject(s)
Cylindrospermopsis/enzymology , Polyketide Synthases/metabolism , Saxitoxin/biosynthesis , Molecular Structure , Polyketide Synthases/chemistry , Saxitoxin/chemistryABSTRACT
Natural product biosynthetic pathways contain a plethora of enzymatic tools to carry out difficult biosynthetic transformations. Here, we discover an unusual mononuclear iron-dependent methyltransferase that acts in the initiation steps of apratoxin A biosynthesis (AprA MT1). Fe3+-replete AprA MT1 catalyzes one or two methyl transfer reactions on the substrate malonyl-ACP (acyl carrier protein), whereas Co2+, Fe2+, Mn2+, and Ni2+ support only a single methyl transfer. MT1 homologues exist within the "GNAT" (GCN5-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryl, or pivaloyl starter units. GNAT domains are thought to catalyze decarboxylation of malonyl-CoA and acetyl transfer to a carrier protein. In AprA, the GNAT domain lacks both decarboxylation and acyl transfer activity. A crystal structure of the AprA MT1-GNAT di-domain with bound Mn2+, malonate, and the methyl donor S-adenosylmethionine (SAM) reveals that the malonyl substrate is a bidentate metal ligand, indicating that the metal acts as a Lewis acid to promote methylation of the malonyl α-carbon. The GNAT domain is truncated relative to functional homologues. These results afford an expanded understanding of MT1-GNAT structure and activity and permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.
Subject(s)
Depsipeptides/biosynthesis , Iron/metabolism , Methyltransferases/metabolism , Polyketides/chemistry , Depsipeptides/chemistry , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Methyltransferases/classification , Methyltransferases/genetics , Models, Molecular , Molecular Structure , Polyketides/metabolism , Protein Conformation , Protein DomainsABSTRACT
Hydrogen sulfide (H2S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H2S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDO and rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO-rhodanese fusion (PRF) proteins in sulfur metabolism. Herein, we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.