Multi-omic based production strain improvement (MOBpsi) for bio-manufacturing of toxic chemicals.

Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations. As a case study, MOBpsi was applied to improve the performance of an Escherichia coli cell factory producing the commodity chemical styrene. Styrene can be bio-manufactured from phenylalanine via an engineered pathway comprised of the enzymes phenylalanine ammonia lyase and ferulic acid decarboxylase. The toxicity, hydrophobicity, and volatility of styrene combine to make bio-production challenging. Previous attempts to create styrene tolerant E. coli strains by targeted genetic interventions have met with modest success. Application of MOBpsi identified new potential targets for improving performance, resulting in two host strains (E. coli NST74ΔaaeA and NST74ΔaaeA cpxPo) with increased styrene production. The best performing re-engineered chassis, NST74ΔaaeA cpxPo, produced ∼3 × more styrene and exhibited increased viability in fed-batch fermentations. Thus, this case study demonstrates the utility of MOBpsi as a systematic tool for improving the bio-manufacturing of toxic chemicals.

Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b.

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.

Decoration of the enterococcal polysaccharide antigen EPA is essential for virulence, cell surface charge and interaction with effectors of the innate immune system.

Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires a complex network of transcriptional regulators and several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.

Regional conformational flexibility couples substrate specificity and scissile phosphate diester selectivity in human flap endonuclease 1.

Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are disordered without substrate. Disorder within the arch explains how 5'-flaps can pass under it. NMR and single-molecule FRET data show a shift in the conformational ensemble in the arch and loop region upon addition of DNA. Furthermore, the addition of divalent metal ions to the active site of the hFEN1-DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.

The Polycystin-1, Lipoxygenase, and α-Toxin Domain Regulates Polycystin-1 Trafficking.

Mutations in polycystin-1 (PC1) give rise to autosomal dominant polycystic kidney disease, an important and common cause of kidney failure. Despite its medical importance, the function of PC1 remains poorly understood. Here, we investigated the role of the intracellular polycystin-1, lipoxygenase, and α-toxin (PLAT) signature domain of PC1 using nuclear magnetic resonance, biochemical, cellular, and in vivo functional approaches. We found that the PLAT domain targets PC1 to the plasma membrane in polarized epithelial cells by a mechanism involving the selective binding of the PLAT domain to phosphatidylserine and L-α-phosphatidylinositol-4-phosphate (PI4P) enriched in the plasma membrane. This process is regulated by protein kinase A phosphorylation of the PLAT domain, which reduces PI4P binding and recruits ß-arrestins and the clathrin adaptor AP2 to trigger PC1 internalization. Our results reveal a physiological role for the PC1-PLAT domain in renal epithelial cells and suggest that phosphorylation-dependent internalization of PC1 is closely linked to its function in renal development and homeostasis.

Ferrous Iron Binding Key to Mms6 Magnetite Biomineralisation: A Mechanistic Study to Understand Magnetite Formation Using pH Titration and NMR Spectroscopy.

Formation of magnetite nanocrystals by magnetotactic bacteria is controlled by specific proteins which regulate the particles' nucleation and growth. One such protein is Mms6. This small, amphiphilic protein can self-assemble and bind ferric ions to aid in magnetite formation. To understand the role of Mms6 during in vitro iron oxide precipitation we have performed in situ pH titrations. We find Mms6 has little effect during ferric salt precipitation, but exerts greatest influence during the incorporation of ferrous ions and conversion of this salt to mixed-valence iron minerals, suggesting Mms6 has a hitherto unrecorded ferrous iron interacting property which promotes the formation of magnetite in ferrous-rich solutions. We show ferrous binding to the DEEVE motif within the C-terminal region of Mms6 by NMR spectroscopy, and model these binding events using molecular simulations. We conclude that Mms6 functions as a magnetite nucleating protein under conditions where ferrous ions predominate.

Near attack conformers dominate ß-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate.

Experimental observations of fluoromagnesate and fluoroaluminate complexes of ß-phosphoglucomutase (ß-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of ß-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of ß-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.

Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF-3 rather than by phosphoranes.

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of beta-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO-3 , but is MgF-3 (trifluoromagnesate). Although MgF-3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19F NMR. Measurements on three beta-PGM-MgF-3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F...H-N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the beta-PGM-MgF--3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF--3 model of near-transition state conformations.

Improved methodology for protein NMR structure calculation using hydrogen bond restraints and ANSURR validation: The SH2 domain of SH2B1.

Protein structures calculated using NMR data are less accurate and less well-defined than they could be. Here we use the program ANSURR to show that this deficiency is at least in part due to a lack of hydrogen bond restraints. We describe a protocol to introduce hydrogen bond restraints into the structure calculation of the SH2 domain from SH2B1 in a systematic and transparent way and show that the structures generated are more accurate and better defined as a result. We also show that ANSURR can be used as a guide to know when the structure calculation is good enough to stop.

Loss of Residues 119-136, Including the First ß-strand of Human Prion Protein, Generates an Aggregation-competent Partially "Open" Form.

In prion replication, the cellular form of prion protein (PrPC) must undergo a full conformational transition to its disease-associated fibrillar form. Transmembrane forms of PrP have been implicated in this structural conversion. The cooperative unfolding of a structural core in PrPC presents a substantial energy barrier to prion formation, with membrane insertion and detachment of parts of PrP presenting a plausible route to its reduction. Here, we examined the removal of residues 119-136 of PrP, a region which includes the first ß-strand and a substantial portion of the conserved hydrophobic region of PrP, a region which associates with the ER membrane, on the structure, stability and self-association of the folded domain of PrPC. We see an "open" native-like conformer with increased solvent exposure which fibrilises more readily than the native state. These data suggest a stepwise folding transition, which is initiated by the conformational switch to this "open" form of PrPC.

When simple sequence comparison fails: the cryptic case of the shared domains of the bacterial replication initiation proteins DnaB and DnaD.

DnaD and DnaB are essential DNA-replication-initiation proteins in low-G+C content Gram-positive bacteria. Here we use sensitive Hidden Markov Model-based techniques to show that the DnaB and DnaD proteins share a common structure that is evident across all their structural domains, termed DDBH1 and DDBH2 (DnaD DnaB Homology 1 and 2). Despite strong sequence divergence, many of the DNA-binding and oligomerization properties of these domains have been conserved. Although eluding simple sequence comparisons, the DDBH2 domains share the only strong sequence motif; an extremely highly conserved YxxxIxxxW sequence that contributes to DNA binding. Sequence alignments of DnaD alone fail to identify another key part of the DNA-binding module, since it includes a poorly conserved sequence, a solvent-exposed and somewhat unstable helix and a mobile segment. We show by NMR, in vitro mutagenesis and in vivo complementation experiments that the DNA-binding module of Bacillus subtilis DnaD comprises the YxxxIxxxW motif, the unstable helix and a portion of the mobile region, the latter two being essential for viability. These structural insights lead us to a re-evaluation of the oligomerization and DNA-binding properties of the DnaD and DnaB proteins.

1H, 13C, and 15N resonance assignments of a conserved putative cell wall binding domain from Enterococcus faecalis.

Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence.

An Enzyme with High Catalytic Proficiency Utilizes Distal Site Substrate Binding Energy to Stabilize the Closed State but at the Expense of Substrate Inhibition.

Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use ß-phosphoglucomutase (ßPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. ßPGM catalyzes the isomerization of ß-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. ßPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported ß-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form.

Prioritization of charge over geometry in transition state analogues of a dual specificity protein kinase.

The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using (19)F NMR analysis of MEK6 in complex with tetrafluoroaluminate (AlF(4)(-)), ADP, and p38α MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF(4)(-) moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF(4)(-) complex involving the alternative acceptor residue in p38α (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF(4)(-) species for both acceptor residues, rather than the trigonal bipyramidal AlF(3)(0) previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion.

High-affinity tamoxifen analogues retain extensive positional disorder when bound to calmodulin.

Using a combination of NMR and fluorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High-affinity binding (Kd∼300 nM) saturates with a 2:1 idoxifene:CaM complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower-affinity antagonists TFP, J-8, and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular nuclear Overhauser effect (NOE) restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity, allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the 2:1 idoxifene:CaM complex illustrates how high-affinity binding occurs without the loss of extensive positional dynamics.

Transition state analogue structures of human phosphoglycerate kinase establish the importance of charge balance in catalysis.

Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within the transition state dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures of trifluoromagnesate (MgF(3)(-)) and tetrafluoroaluminate (AlF(4)(-)) complexes of PGK have been determined using X-ray crystallography and (19)F-based NMR methods, revealing the nature of the catalytically relevant state of this archetypal metabolic kinase. Importantly, the side chain of K219, which coordinates the alpha-phosphate group in previous ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphoryl group. In line with the dominance of charge balance in transition state organization, the substitution K219A induces a corresponding reduction in charge in the bound aluminum fluoride species, which changes to a trifluoroaluminate (AlF(3)(0)) complex. The AlF(3)(0) moiety retains the octahedral geometry observed within AlF(4)(-) TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF(3)(0) complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF(3)(-).

Allomorphy as a mechanism of post-translational control of enzyme activity.

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. ß-phosphoglucomutase (ßPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of ßPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In ßPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate ß-glucose 1,6-bisphosphate, whose concentration depends on the ß-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

Structural effects of the highly protective V127 polymorphism on human prion protein.

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP ß-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the ß2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.

MgF(3)(-) and alpha-galactose 1-phosphate in the active site of beta-phosphoglucomutase form a transition state analogue of phosphoryl transfer.

(19)F-based NMR analysis and hydrogen/deuterium primary isotope shifts establish the formation of a highly populated solution-state trigonal bipyramidal complex involving beta-phosphoglucomutase (beta-PGM), alpha-galactose 1-phosphate (alphaGal1P), and trifluoromagnesate (MgF(3)(-)), PGM-MgF(3)-alphaGal1P, that is a transition state analogue for phosphoryl transfer. Full backbone resonance assignment of the protein shows that its structure is in the closed conformation required for catalytic activity and is closely related to the corresponding complex with glucose 6-phosphate, which we have recently identified using NMR analysis in solution and X-ray crystallography in the solid state. The previous identification of three structural waters in a PGM-alphaGal1P binary substrate complex had indicated that, in the presence of alphaGal1P, magnesium ions, and fluoride, beta-PGM should indeed form a PGM-MgF(3)-alphaGal1P-TSA complex whereas, in the solid-state, apparently it did not. This cast doubt on the validity of the interpretation of MgF(3)(-) complexes. The present work establishes that, in solution, the expectation that a PGM-MgF(3)-alphaGal1P-TSA complex should readily form is fulfilled. These results thus refute the final evidence used to claim that the trigonal bipyramidal species observed in some solid-state structures of complexes involving beta-PGM are pentaoxyphosphorane intermediates.

1H, 15N and 13C backbone resonance assignments of the P146A variant of ß-phosphoglucomutase from Lactococcus lactis in its substrate-free form.

ß-Phosphoglucomutase (ßPGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of ß-glucose 1-phosphate and glucose 6-phosphate, via two phosphoryl transfer steps and a ß-glucose 1,6-bisphosphate intermediate. Substrate-free ßPGM is an essential component of the catalytic cycle and an understanding of its dynamics would present significant insights into ßPGM functionality, and enzyme catalysed phosphoryl transfer in general. Previously, 30 residues around the active site of substrate-free ßPGMWT were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report 1H, 15N and 13C backbone resonance assignments of the P146A variant (ßPGMP146A) in its substrate-free form, where the K145-A146 peptide bond adopts a trans conformation in contrast to all crystal structures of ßPGMWT, where the K145-P146 peptide bond is cis. In ßPGMP146A millisecond dynamics are suppressed for all but 17 residues, allowing 92% of backbone resonances to be assigned. Secondary structure predictions using TALOS-N reflect ßPGM crystal structures, and a chemical shift comparison between substrate-free ßPGMP146A and ßPGMWT confirms that the solution conformations are very similar, except for the D137-A147 loop. Hence, the isomerisation state of the 145-146 peptide bond has little effect on structure but the cis conformation triggers millisecond dynamics in the hinge (V12-T16), the nucleophile (D8) and residues that coordinate the transferring phosphate group (D8 and S114-S116), and the D137-A147 loop (V141-A142 and K145). These millisecond dynamics occur in addition to those for residues involved in coordinating the catalytic MgII ion and the L44-L53 loop responsible for substrate discrimination.