A generalizable protocol for expression and purification of membrane-bound bacterial phosphoglycosyl transferases in liponanoparticles.

Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of difficult membrane proteins directly from cell membranes into native-like liponanoparticles. By leveraging autoinduction, in vivo SUMO tag cleavage, styrene maleic acid co-polymer liponanoparticles (SMALPs), and Strep-Tag purification, we have established a robust workflow for expression and purification of previously unobtainable PGTs. The material generated from this workflow is extremely pure and can be directly visualized by Cryogenic Electron Microscopy (CryoEM). The methods presented here promise to be generalizable to additional membrane proteins recombinantly expressed in E. coli and should be of interest to the greater membrane proteomics community.

Structural and dynamical rationale for fatty acid unsaturation in Escherichia coli.

Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of six Escherichia coli FabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.

Sacrificial Cobalt-Carbon Bond Homolysis in Coenzyme B12 as a Cofactor Conservation Strategy.

A sophisticated intracellular trafficking pathway in humans is used to tailor vitamin B12 into its active cofactor forms, and to deliver it to two known B12-dependent enzymes. Herein, we report an unexpected strategy for cellular retention of B12, an essential and reactive cofactor. If methylmalonyl-CoA mutase is unavailable to accept the coenzyme B12 product of adenosyltransferase, the latter catalyzes homolytic scission of the cobalt-carbon bond in an unconventional reversal of the nucleophilic displacement reaction that was used to make it. The resulting homolysis product binds more tightly to adenosyltransferase than does coenzyme B12, facilitating cofactor retention. We have trapped, and characterized spectroscopically, an intermediate in which the cobalt-carbon bond is weakened prior to being broken. The physiological relevance of this sacrificial catalytic activity for cofactor retention is supported by the significantly lower coenzyme B12 concentration in patients with dysfunctional methylmalonyl-CoA mutase but normal adenosyltransferase activity.

Protein-protein interactions in "cis-AT" polyketide synthases.

Covering: up to the end of 2018 Polyketides are a valuable source of bioactive and clinically important molecules. The biosynthesis of these chemically complex molecules has led to the discovery of equally complex polyketide synthase (PKS) pathways. Crystallography has yielded snapshots of individual catalytic domains, di-domains, and multi-domains from a variety of PKS megasynthases, and cryo-EM studies have provided initial views of a PKS module in a series of defined biochemical states. Here, we review the structural and biochemical results that shed light on the protein-protein interactions critical to catalysis by PKS systems with an embedded acyltransferase. Interactions include those that occur both within and between PKS modules, as well as with accessory enzymes.

Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis.

Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels, and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during polyketide biosynthesis through the dehydration of the nascent polyketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both cis- and trans-olefins, providing an unrivaled model system for polyketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized in vitro using synthetic substrates, and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the all-trans-trienoate-forming CurK-DH, whose kcat of 72 s-1 is more than 3 orders of magnitude greater than that of any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH DHs on the basis of incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ-DH to elucidate substrate-enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme-substrate interactions essential for the formation of olefins in polyketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology.

Annotation of proteins of unknown function: initial enzyme results.

Working with a combination of ProMOL (a plugin for PyMOL that searches a library of enzymatic motifs for local structural homologs), BLAST and Pfam (servers that identify global sequence homologs), and Dali (a server that identifies global structural homologs), we have begun the process of assigning functional annotations to the approximately 3,500 structures in the Protein Data Bank that are currently classified as having "unknown function". Using a limited template library of 388 motifs, over 500 promising in silico matches have been identified by ProMOL, among which 65 exceptionally good matches have been identified. The characteristics of the exceptionally good matches are discussed.

Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase.

Metabolic engineering of polyketide synthase (PKS) pathways represents a promising approach to natural products discovery. The dehydratase (DH) domains of PKSs, which generate an α,ß-unsaturated bond through a dehydration reaction, have been poorly studied compared with other domains, likely because of the simple nature of the chemical reaction they catalyze and the lack of a convenient assay to measure substrate turnover. Herein we report the first steady-state kinetic analysis of a PKS DH domain employing LC-MS/MS analysis for product quantitation. PikDH2 was selected as a model DH domain. Its substrate specificity and mechanism were interrogated with a systematic series of synthetic triketide substrates containing a nonhydrolyzable thioether linkage as well as by site-directed mutagenesis, evaluation of the pH dependence of the catalytic efficiency (V(max)/K(M)), and kinetic characterization of a mechanism-based inhibitor. These studies revealed that PikDH2 converts d-alcohol substrates to trans-olefin products. The reaction is reversible with equilibrium constants ranging from 1.2 to 2. Moreover, the enzyme activity is robust, and PikDH2 was used on a preparative scale for the chemoenzymatic synthesis of unsaturated triketide products. PikDH2 was shown to possess remarkably strict substrate specificity and is unable to turn over substrates that are epimeric at the ß-, γ-, or δ-position. We also demonstrated that PikDH2 has a key ionizable group with a pK(a) of 7.0 and can be irreversibly inactivated through covalent modification by a mechanism-based inhibitor, which provides a foundation for future structural studies to elucidate substrate-protein interactions.

Estimation of protein function using template-based alignment of enzyme active sites.

BACKGROUND: The accumulation of protein structural data occurs more rapidly than it can be characterized by traditional laboratory means. This has motivated widespread efforts to predict enzyme function computationally. The most useful/accurate strategies employed to date are based on the detection of motifs in novel structures that correspond to a specific function. Functional residues are critical components of predictively useful motifs. We have implemented a novel method, to complement current approaches, which detects motifs solely on the basis of distance restraints between catalytic residues. RESULTS: ProMOL is a plugin for the PyMOL molecular graphics environment that can be used to create active site motifs for enzymes. A library of 181 active site motifs has been created with ProMOL, based on definitions published in the Catalytic Site Atlas (CSA). Searches with ProMOL produce better than 50% useful Enzyme Commission (EC) class suggestions for level 1 searches in EC classes 1, 4 and 5, and produce some useful results for other classes. 261 additional motifs automatically translated from Jonathan Barker's JESS motif set [Bioinformatics 19:1644-1649, 2003] and a set of NMR motifs is under development. Alignments are evaluated by visual superposition, Levenshtein distance and root-mean-square deviation (RMSD) and are reasonably consistent with related search methods. CONCLUSION: The ProMOL plugin for PyMOL provides ready access to template-based local alignments. Recent improvements to ProMOL, including the expanded motif library, RMSD calculations and output selection formatting, have greatly increased the program's usability and speed, and have improved the way that the results are presented.

Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle.

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.

Proteome-Wide Bioinformatic Annotation and Functional Validation of the Monotopic Phosphoglycosyl Transferase Superfamily.

Phosphoglycosyl transferases (PGTs) are membrane proteins that initiate glycoconjugate biosynthesis by transferring a phospho-sugar moiety from a soluble nucleoside diphosphate sugar to a membrane-embedded polyprenol phosphate acceptor. The centrality of PGTs in complex glycan assembly and the current lack of functional information make these enzymes high-value targets for biochemical investigation. In particular, the small monotopic PGT family is exclusively bacterial and represents the minimal functional unit of the monotopic PGT superfamily. Here, we combine a sequence similarity network (SSN) analysis with a generalizable, luminescence-based activity assay to probe the substrate specificity of this family of monoPGTs in a bacterial cell-membrane fraction. This strategy allows us to identify specificity on a far more significant scale than previously achievable and correlate preferred substrate specificities with predicted structural differences within the conserved monoPGT fold. Finally, we present the proof-of-concept for a small-scale inhibitor screen (eight nucleoside analogs) with four monoPGTs of diverse substrate specificity, thus building a foundation for future inhibitor discovery initiatives.

Co-conserved sequence motifs are predictive of substrate specificity in a family of monotopic phosphoglycosyl transferases.

Monotopic phosphoglycosyl transferases (monoPGTs) are an expansive superfamily of enzymes that catalyze the first membrane-committed step in the biosynthesis of bacterial glycoconjugates. MonoPGTs show a strong preference for their cognate nucleotide diphospho-sugar (NDP-sugar) substrates. However, despite extensive characterization of the monoPGT superfamily through previous development of a sequence similarity network comprising >38,000 nonredundant sequences, the connection between monoPGT sequence and NDP-sugar substrate specificity has remained elusive. In this work, we structurally characterize the C-terminus of a prototypic monoPGT for the first time and show that 19 C-terminal residues play a significant structural role in a subset of monoPGTs. This new structural information facilitated the identification of co-conserved sequence "fingerprints" that predict NDP-sugar substrate specificity for this subset of monoPGTs. A Hidden Markov model was generated that correctly assigned the substrate of previously unannotated monoPGTs. Together, these structural, sequence, and biochemical analyses have delivered new insight into the determinants guiding substrate specificity of monoPGTs and have provided a strategy for assigning the NDP-sugar substrate of a subset of enzymes in the superfamily that use UDP-di-N-acetyl bacillosamine. Moving forward, this approach may be applied to identify additional sequence motifs that serve as fingerprints for monoPGTs of differing UDP-sugar substrate specificity.

Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle.

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from Salmonella enterica (LT2) O-antigen biosynthesis without detergent solubilization from the lipid bilayer. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for oligomerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying small membrane proteins embedded in liponanoparticles beyond PGTs.

Molecular Basis for Olefin Rearrangement in the Gephyronic Acid Polyketide Synthase.

Polyketide synthases (PKS) are a rich source of natural products of varied chemical composition and biological significance. Here, we report the characterization of an atypical dehydratase (DH) domain from the PKS pathway for gephyronic acid, an inhibitor of eukaryotic protein synthesis. Using a library of synthetic substrate mimics, the reaction course, stereospecificity, and tolerance to non-native substrates of GphF DH1 are probed via LC-MS analysis. Taken together, the studies establish GphF DH1 as a dual-function dehydratase/isomerase that installs an odd-to-even double bond and yields a product consistent with the isobutenyl terminus of gephyronic acid. The studies also reveal an unexpected C2 epimerase function in catalytic turnover with the native substrate. A 1.55-Å crystal structure of GphF DH1 guided mutagenesis experiments to elucidate the roles of key amino acids in the multistep DH1 catalysis, identifying critical functions for leucine and tyrosine side chains. The mutagenesis results were applied to add a secondary isomerase functionality to a nonisomerizing DH in the first successful gain-of-function engineering of a PKS DH. Our studies of GphF DH1 catalysis highlight the versatility of the DH active site and adaptation for a specific catalytic outcome with a specific substrate.

Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of ß-processing domains via diffusible, synthetic substrates.

Polyketide synthase (PKS) ß-processing domains are responsible for much of the stereochemical complexity of polyketide natural products. Although the importance of ß-processing domains has been well noted and significantly explored, key stereochemical details pertaining to cryptic stereochemistry and the impact of remote stereogenic centers have yet to be fully discerned. To uncover the inner workings of ketoreductases (KR) and dehydratases (DH) from the tylosin pathway a didomain composed of TylDH3-KR3 was recombinantly expressed and interrogated with full-length tetraketide substrates to probe the impact of vicinal and distal stereochemistry. In vitro product isolation analysis revealed the products of the cryptic KR as d-alcohols and of the DH as trans-olefins. Steady-state kinetic analysis of the dehydration reaction demonstrated a strict stereochemical tolerance at the ß-position as d-configured substrates were processed more than 100 times more efficiently than l-alcohols. Unexpectedly, the kcat/KM values were diminished 14- to 45-fold upon inversion of remote ε- and ζ-stereocenters. This stereochemical discrimination is predicted to be driven by a combination of allylic A1,3 strain that likely disfavors binding of the ε-epimer and a loss of electrostatic interactions with the ζ-epimer. Our results strongly suggest that dehydratases may play a role in refining the stereochemical outcomes of preceding modules through their substrate stereospecificity, honing the configurational purity of the final PKS product.

Low-impact pelvic fractures in the emergency department.

OBJECTIVE: We examined the records of patients presenting to the emergency department (ED) with low-impact pelvic fractures. We describe frequency, demographics, management and patient outcomes in terms of ambulatory ability, living independence and mortality. METHODS: Patients treated for a pelvic fracture over a 2-year period in Kingston, Ont., were identified. We performed a retrospective hospital record review to distinguish high- versus low-impact injury mechanisms, and to characterize the injury event, ED management and outcomes for patients with low-impact fractures. RESULTS: Of 132 pelvic fractures identified, 77 were low-impact fractures. Patients were predominantly women (82%) with a mean age of 81 years; 96% had some pre-existing medical comorbidity. The pubic rami were most commonly involved (86%). The median length of stay in the ED was 9.4 hours. Twenty-five patients (32%) were admitted to hospital. Ten patients had surgical stabilization, mostly of the acetabulum. Five patients died in hospital, 4 from pneumonia and 1 from myocardial infarction. Eight additional patients died within 1 year of injury. At discharge, only 18% lived independently and 16% walked without aids versus 42% and 38%, respectively, before injury. CONCLUSION: Low-impact pelvic fractures affect predominantly elderly women with pre-existing comorbidities. A substantial amount of time and resources in the ED are used during the workup of these patients and while awaiting their disposition from the ED. These injuries are important because they affect independence and seem associated with an increased risk of death.