Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids.

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A2, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (Vmax∼15.5 µmol/min/mg; Km∼83 µM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall-deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.

Oligomeric complexes formed by Redß single strand annealing protein in its different DNA bound states.

Redß is a single strand annealing protein from bacteriophage λ that binds loosely to ssDNA, not at all to pre-formed dsDNA, but tightly to a duplex intermediate of annealing. As viewed by electron microscopy, Redß forms oligomeric rings on ssDNA substrate, and helical filaments on the annealed duplex intermediate. However, it is not clear if these are the functional forms of the protein in vivo. We have used size-exclusion chromatography coupled with multi-angle light scattering, analytical ultracentrifugation and native mass spectrometry (nMS) to characterize the size of the oligomers formed by Redß in its different DNA-bound states. The nMS data, which resolve species with the highest resolution, reveal that Redß forms an oligomer of 12 subunits in the absence of DNA, complexes ranging from 4 to 14 subunits on 38-mer ssDNA, and a much more distinct and stable complex of 11 subunits on 38-mer annealed duplex. We also measure the concentration of Redß in cells active for recombination and find it to range from 7 to 27 µM. Collectively, these data provide new insights into the dynamic nature of the complex on ssDNA, and the more stable and defined complex on annealed duplex.

Crystal structure of the Redß C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction with Escherichia coli single stranded DNA binding protein.

Bacteriophage λ encodes a DNA recombination system that includes a 5'-3' exonuclease (λ Exo) and a single strand annealing protein (Redß). The two proteins form a complex that is thought to mediate loading of Redß directly onto the single-stranded 3'-overhang generated by λ Exo. Here, we present a 2.3 Å crystal structure of the λ Exo trimer bound to three copies of the Redß C-terminal domain (CTD). Mutation of residues at the hydrophobic core of the interface disrupts complex formation in vitro and impairs recombination in vivo. The Redß CTD forms a three-helix bundle with unexpected structural homology to phage λ Orf, a protein that binds to E. coli single-stranded DNA binding protein (SSB) to function as a recombination mediator. Based on this relationship, we found that Redß binds to full-length SSB, and to a peptide corresponding to its nine C-terminal residues, in an interaction that requires the CTD. These results suggest a dual role of the CTD, first in binding to λ Exo to facilitate loading of Redß directly onto the initial single-stranded DNA (ssDNA) at a 3'-overhang, and second in binding to SSB to facilitate annealing of the overhang to SSB-coated ssDNA at the replication fork.

Mutational Analysis of Redß Single Strand Annealing Protein: Roles of the 14 Lysine Residues in DNA Binding and Recombination In Vivo.

Redß is a 261 amino acid protein from bacteriophage λ that promotes a single-strand annealing (SSA) reaction for repair of double-stranded DNA (dsDNA) breaks. While there is currently no high-resolution structure available for Redß, models of its DNA binding domain (residues 1-188) have been proposed based on homology with human Rad52, and a crystal structure of its C-terminal domain (CTD, residues 193-261), which binds to λ exonuclease and E. coli single-stranded DNA binding protein (SSB), has been determined. To evaluate these models, the 14 lysine residues of Redß were mutated to alanine, and the variants tested for recombination in vivo and DNA binding and annealing in vitro. Most of the lysines within the DNA binding domain, including K36, K61, K111, K132, K148, K154, and K172, were found to be critical for DNA binding in vitro and recombination in vivo. By contrast, none of the lysines within the CTD, including K214, K245, K251, K253, and K258 were required for DNA binding in vitro, but two, K214 and K253, were critical for recombination in vivo, likely due to their involvement in binding to SSB. K61 was identified as a residue that is critical for DNA annealing, but not for initial ssDNA binding, suggesting a role in binding to the second strand of DNA incorporated into the complex. The K148A variant, which has previously been shown to be defective in oligomer formation, had the lowest affinity for ssDNA, and was the only variant that was completely non-cooperative, suggesting that ssDNA binding is coupled to oligomerization.

The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase.

Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 µmol/min/mg protein and a Km of 45 µM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.

Mutant poisoning demonstrates a nonsequential mechanism for digestion of double-stranded DNA by λ exonuclease trimers.

λ Exonuclease (λexo) is a highly processive 5'-3' exonuclease that binds double-stranded DNA (dsDNA) ends and digests the 5'-strand into mononucleotides. The enzyme forms a toroidal homotrimer with a central tapered channel for tracking along the DNA. During catalysis, dsDNA enters the open end of the channel, and the 5'-strand is digested at one of the three active sites. It is currently not known if λexo uses a sequential mechanism, in which the DNA moves from one active site to the next around the trimer for each round of catalysis or a nonsequential mechanism, in which the DNA locks onto a single active site for multiple rounds. To understand how λexo uses its three active sites, we used a mutant poisoning approach, in which a 6xHis-tagged K131A inactive mutant of λexo was mixed with untagged wild type (WT) to form hybrid trimers. Nickel-spin pull-down analysis confirmed complete subunit exchange after 1 h at 37 °C. Exonuclease assays revealed an approximately linear decrease in activity with increasing fraction of mutant, as expected for a nonsequential mechanism. By fitting the observed rates of digestion to a simple mathematical model, the individual rates of the two hybrid species of trimer were determined. This analysis showed that trimers containing only one or two WT subunits contribute significantly to the observed activity, in further agreement with a nonsequential mechanism. Finally, purification of hybrid trimer mixtures by Ni-spin chromatography, to remove the contribution from fully WT trimers, also resulted in significant levels of activity, again consistent with a nonsequential mechanism.

A Structure-Activity Analysis for Probing the Mechanism of Processive Double-Stranded DNA Digestion by λ Exonuclease Trimers.

λ exonuclease (λexo) is an ATP-independent 5'-to-3' exonuclease that binds to double-stranded DNA (dsDNA) ends and processively digests the 5'-strand into mononucleotides. The crystal structure of λexo revealed that the enzyme forms a ring-shaped homotrimer with a central funnel-shaped channel for tracking along the DNA. On the basis of this structure, it was proposed that dsDNA enters the open end of the channel, the 5'-strand is digested at one of the three active sites, and the 3'-strand passes through the narrow end of the channel to emerge out the back. This model was largely confirmed by the structure of the λexo-DNA complex, which further revealed that the enzyme unwinds the DNA by 2 bp prior to cleavage, to thread the 5'-end of the DNA into the active site. On the basis of this structure, an "electrostatic ratchet" model was proposed, in which the enzyme uses a hydrophobic wedge to insert into the base pairs to unwind the DNA, a two-metal mechanism for nucleotide hydrolysis, a positively charged pocket to bind to the terminal 5'-phosphate generated after each round of cleavage, and an arginine residue (Arg-45) to bind to the minor groove of the downstream end of the DNA. To test this model, in this study we have determined the effects of 11 structure-based mutations in λexo on DNA binding and exonuclease activities in vitro, and on DNA recombination in vivo. The results are largely consistent with the model for the mechanism that was proposed on the basis of the structure and provide new insights into the roles of particular residues of the protein in promoting the reaction. In particular, a key role for Arg-45 in DNA binding is revealed.

Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion.

Escherichia coli Exonuclease I (ExoI) digests single-stranded DNA (ssDNA) in the 3'-5' direction in a highly processive manner. The crystal structure of ExoI, determined previously in the absence of DNA, revealed a C-shaped molecule with three domains that form a central positively charged groove. The active site is at the bottom of the groove, while an extended loop, proposed to encircle the DNA, crosses over the groove. Here, we present crystal structures of ExoI in complex with four different ssDNA substrates. The structures all have the ssDNA bound in essentially the predicted manner, with the 3'-end in the active site and the downstream end under the crossover loop. The central nucleotides of the DNA form a prominent bulge that contacts the SH3-like domain, while the nucleotides at the downstream end of the DNA form extensive interactions with an 'anchor' site. Seven of the complexes are similar to one another, but one has the ssDNA bound in a distinct conformation. The highest-resolution structure, determined at 1.95 Å, reveals an Mg(2+) ion bound to the scissile phosphate in a position corresponding to Mg(B) in related two-metal nucleases. The structures provide new insights into the mechanism of processive digestion that will be discussed.

Crystal structure of λ exonuclease in complex with DNA and Ca(2+).

Bacteriophage λ exonuclease (λexo) is a ring-shaped homotrimer that resects double-stranded DNA ends in the 5'-3' direction to generate a long 3'-overhang that is a substrate for recombination. λexo is a member of the type II restriction endonuclease-like superfamily of proteins that use a Mg(2+)-dependent mechanism for nucleotide cleavage. A previous structure of λexo in complex with DNA and Mg(2+) was determined using a nuclease defective K131A variant to trap a stable complex. This structure revealed the detailed coordination of the two active site Mg(2+) ions but did not show the interactions involving the side chain of the conserved active site Lys-131 residue. Here, we have determined the crystal structure of wild-type (WT) λexo in complex with the same DNA substrate, but in the presence of Ca(2+) instead of Mg(2+). Surprisingly, there is only one Ca(2+) bound in the active site, near the position of Mg(A) in the structure with Mg(2+). The scissile phosphate is displaced by 2.2 Å relative to its position in the structure with Mg(2+), and the network of interactions involving the attacking water molecule is broken. Thus, the structure does not represent a catalytic configuration. However, the crystal structure does show clear electron density for the side chain of Lys-131, which is held in place by interactions with Gln-157 and Glu-129. By combining the K131A-Mg(2+) and WT-Ca(2+) structures, we constructed a composite model to show the likely interactions of Lys-131 during catalysis. The implications with regard to the catalytic mechanism are discussed.

Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases.

The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >10(5)-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >10(5)-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3-18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A cocrystal structure of PTP1B bound with a nephrin pY(1193) peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities, and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic activities with respect to PTP in vivo substrate specificity and biological functions are discussed.

Crystal structures of lambda exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity.

The λ exonuclease is an ATP-independent enzyme that binds to dsDNA ends and processively digests the 5'-ended strand to form 5' mononucleotides and a long 3' overhang. The crystal structure of λ exonuclease revealed a toroidal homotrimer with a central funnel-shaped channel for tracking along the DNA, and a mechanism for processivity based on topological linkage of the trimer to the DNA was proposed. Here, we have determined the crystal structure of λ exonuclease in complex with DNA at 1.88-Å resolution. The structure reveals that the enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5'-ended strand insert into the active site of one subunit of the trimer, while the 3'-ended strand passes through the central channel to emerge out the back of the trimer. Unwinding of the DNA is facilitated by several apolar residues, including Leu78, that wedge into the base pairs at the single/double-strand junction to form favorable hydrophobic interactions. The terminal 5' phosphate of the DNA binds to a positively charged pocket buried at the end of the active site, while the scissile phosphate bridges two active site Mg(2+) ions. Our data suggest a mechanism for processivity in which wedging of Leu78 and other apolar residues into the base pairs of the DNA restricts backward movement, whereas attraction of the 5' phosphate to the positively charged pocket drives forward movement of the enzyme along the DNA at each cycle of the reaction. Thus, processivity of λ exonuclease operates not only at the level of the trimer, but also at the level of the monomer.

Structural Basis for the Interaction of Redß Single-Strand Annealing Protein with Escherichia coli Single-Stranded DNA-Binding Protein.

Redß is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease (λ-exo), Redß is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redß can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redß with E. coli single-stranded DNA-binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redß and SSB involves the C-terminal domain (CTD) of Redß and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redß CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redß and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redß-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology.

Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose-lysine.

Amadori rearrangement products are stable sugar-amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose-lysine (F-Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F-Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6-phosphofructose-lysine (6-P-F-Lys). FrlB, a deglycase, then converts 6-P-F-Lys to L-lysine and glucose-6-phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8-Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6-P-F-Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine-6-phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB-6-P-F-Lys on GlmS-fructose-6-phosphate structures revealed parallels in their active-site arrangement and guided our selection of seven putative active-site residues in FrlB for site-directed mutagenesis. Activity assays with eight recombinant single-substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface-induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x-ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure-function and mechanistic studies of enzymes.

Structure of a RecT/Redß family recombinase in complex with a duplex intermediate of DNA annealing.

Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA). These proteins are apparently related to RAD52, the primary human SSA protein. The best studied protein, Redß from bacteriophage λ, binds weakly to ssDNA, not at all to dsDNA, but tightly to a duplex intermediate of annealing formed when two complementary DNA strands are added to the protein sequentially. We used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a Redß homolog from a prophage of Listeria innocua in complex with two complementary 83mer oligonucleotides. The structure reveals a helical protein filament bound to a DNA duplex that is highly extended and unwound. Native mass spectrometry confirms that the complex seen by cryo-EM is the predominant species in solution. The protein shares a common core fold with RAD52 and a similar mode of ssDNA-binding. These data provide insights into the mechanism of protein-catalyzed SSA.

Crystal structure of E. coli RecE protein reveals a toroidal tetramer for processing double-stranded DNA breaks.

Escherichia coli RecE protein is part of the classical RecET recombination system that has recently been used in powerful new methods for genetic engineering. RecE binds to free double-stranded DNA (dsDNA) ends and processively digests the 5'-ended strand to form 5'-mononucleotides and a 3'-overhang that is a substrate for single strand annealing promoted by RecT. Here, we report the crystal structure of the C-terminal nuclease domain of RecE at 2.8 A resolution. RecE forms a toroidal tetramer with a central tapered channel that is wide enough to bind dsDNA at one end, but is partially plugged at the other end by the C-terminal segment of the protein. Four narrow tunnels, one within each subunit of the tetramer, lead from the central channel to the four active sites, which lie about 15 A from the channel. The structure, combined with mutational studies, suggests a mechanism in which dsDNA enters through the open end of the central channel, the 5'-ended strand passes through a tunnel to access one of the four active sites, and the 3'-ended strand passes through the plugged end of the channel at the back of the tetramer.

Probing the catalytic mechanism of S-ribosylhomocysteinase (LuxS) with catalytic intermediates and substrate analogues.

S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). The catalytic mechanism of LuxS comprises three distinct reaction steps. The first step involves carbonyl migration from the C1 carbon of ribose to C2 and the formation of a 2-ketone intermediate. The second step shifts the C=O group from the C2 to C3 position to produce a 3-ketone intermediate. In the final step, the 3-ketone intermediate undergoes a beta-elimination reaction resulting in the cleavage of the thioether bond. In this work, the 3-ketone intermediate was chemically synthesized and shown to be chemically and kinetically competent in the LuxS catalytic pathway. Substrate analogues halogenated at the C3 position of ribose were synthesized and reacted as time-dependent inhibitors of LuxS. The time dependence was caused by enzyme-catalyzed elimination of halide ions. Examination of the kinetics of halide release and decay of the 3-ketone intermediate catalyzed by wild-type and mutant LuxS enzymes revealed that Cys-84 is the general base responsible for proton abstraction in the three reaction steps, whereas Glu-57 likely facilitates substrate binding and proton transfer during catalysis.

Structure and mechanism of the Red recombination system of bacteriophage λ.

While much of this volume focuses on mammalian DNA repair systems that are directly involved in genome stability and cancer, it is important to still be mindful of model systems from prokaryotes. Herein we review the Red recombination system of bacteriophage λ, which consists of an exonuclease for resecting dsDNA ends, and a single-strand annealing protein (SSAP) for binding the resulting 3'-overhang and annealing it to a complementary strand. The genetics and biochemistry of Red have been studied for over 50 years, in work that has laid much of the foundation for understanding DNA recombination in higher eukaryotes. In fact, the Red exonuclease (λ exo) is homologous to Dna2, a nuclease involved in DNA end-resection in eukaryotes, and the Red annealing protein (Redß) is homologous to Rad52, the primary SSAP in eukaryotes. While eukaryotic recombination involves an elaborate network of proteins that is still being unraveled, the phage systems are comparatively simple and streamlined, yet still encompass the fundamental features of recombination, namely DNA end-resection, homologous pairing (annealing), and a coupling between them. Moreover, the Red system has been exploited in powerful methods for bacterial genome engineering that are important for functional genomics and systems biology. However, several mechanistic aspects of Red, particularly the action of the annealing protein, remain poorly understood. This review will focus on the proteins of the Red recombination system, with particular attention to structural and mechanistic aspects, and how the lessons learned can be applied to eukaryotic systems.

Integrated Use of Biochemical, Native Mass Spectrometry, Computational, and Genome-Editing Methods to Elucidate the Mechanism of a Salmonella deglycase.

Salmonellais a foodborne pathogen that causes annually millions of cases of salmonellosis globally, yet Salmonella-specific antibacterials are not available. During inflammation, Salmonella utilizes the Amadori compound fructose-asparagine (F-Asn) as a nutrient through the successive action of three enzymes, including the terminal FraB deglycase. Salmonella mutants lacking FraB are highly attenuated in mouse models of inflammation due to the toxic build-up of the substrate 6-phosphofructose-aspartate (6-P-F-Asp). This toxicity makes Salmonella FraB an appealing drug target, but there is currently little experimental information about its catalytic mechanism. Therefore, we sought to test our postulated mechanism for the FraB-catalyzed deglycation of 6-P-F-Asp (via an enaminol intermediate) to glucose-6-phosphate and aspartate. A FraB homodimer model generated by RosettaCM was used to build substrate-docked structures that, coupled with sequence alignment of FraB homologs, helped map a putative active site. Five candidate active-site residues-including three expected to participate in substrate binding-were mutated individually and characterized. Native mass spectrometry and ion mobility were used to assess collision cross sections and confirm that the quaternary structure of the mutants mirrored the wild type, and that there are two active sites/homodimer. Our biochemical studies revealed that FraB Glu214Ala, Glu214Asp, and His230Ala were inactive in vitro, consistent with deprotonated-Glu214 and protonated-His230 serving as a general base and a general acid, respectively. Glu214Ala or His230Ala introduced into the Salmonella chromosome by CRISPR/Cas9-mediated genome editing abolished growth on F-Asn. Results from our computational and experimental approaches shed light on the catalytic mechanism of Salmonella FraB and of phosphosugar deglycases in general.

Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators.

Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

Probing the DNA sequence specificity of Escherichia coli RECA protein.

Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5'-GCTGGTGG-3'). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a lambda repressor fragment in the presence of RecA, ADP-AlF4 and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed.