Structural characterization of lytic transglycosylase MltD of Pseudomonas aeruginosa, a target for the natural product bulgecin A.

Natural product bulgecin A potentiates the activity of ß-lactam antibiotics by inhibition of three lytic transglycosylases in Pseudomonas aeruginosa, of which MltD is one. MltD exhibits both endolytic and exolytic reactions in the turnover of the cell-wall peptidoglycan and tolerates the presence or absence of stem peptides in its substrates. The present study reveals structural features of the multimodular MltD, presenting a catalytic module and four cell-wall-binding LysM modules that account for these attributes. Three X-ray structures are reported herein for MltD that disclose one unpredicted LysM module tightly attached to the catalytic domain, whereas the other LysM modules are mobile, and connected to the catalytic domain through long flexible linkers. The formation of crystals depended on the presence of bulgecin A. The expansive active-site cleft is highlighted by the insertion of a helical region, a hallmark of the family 1D of lytic transglycosylases, which was mapped out in a ternary complex of MltD:bulgecinA:chitotetraose, revealing at the minimum the presence of eight subsites (from -4 to +4, with the seat of reaction at subsites -1 and + 1) for binding of sugars of the substrate for the endolytic reaction. The mechanism of the exolytic reaction is revealed in one of the structures, showing how the substrate's terminal anhydro-NAM moiety could be sequestered at subsite +2. Our results provide the structural insight for both the endolytic and exolytic activities of MltD during cell-wall-turnover events.

Catalytic process of anhydro-N-acetylmuramic acid kinase from Pseudomonas aeruginosa.

The bacterial cell envelope is the structure with which the bacterium engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the pairing of hydrolytic ring opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM, and AnmK:ATP:anhNAM) demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. A remarkable X-ray structure for dimeric AnmK sheds light on the precatalytic and postcatalytic ternary complexes. Computational simulations in conjunction with the high-resolution X-ray structures reveal the full catalytic cycle. We further report that a P. aeruginosa strain with disrupted anmK gene is more susceptible to the ß-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.

Amber-codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase.

The 11 lytic transglycosylases of Pseudomonas aeruginosa have overlapping activities in the turnover of the cell-wall peptidoglycan. Rare lipoprotein A (RlpA) is distinct among the 11 by its use of only peptidoglycan lacking peptide stems. The spatial localization of RlpA and its interactome within P. aeruginosa are unknown. We employed suppression of introduced amber codons at sites in the rlpA gene for the introduction of the unnatural-amino-acids Νζ -[(2-azidoethoxy)carbonyl]-l-lysine (compound 1) and Nζ -[[[3-(3-methyl-3H-diazirin-3-yl)propyl]amino]carbonyl]-l-lysine (compound 2). In live P. aeruginosa, full-length RlpA incorporating compound 1 into its sequence was fluorescently tagged using strained-promoted alkyne-azide cycloaddition and examined by fluorescence microscopy. RlpA is present at low levels along the sidewall length of the bacterium, and at higher levels at the nascent septa of replicating bacteria. In intact P. aeruginosa, UV photolysis of full-length RlpA having compound 2 within its sequence generated a transient reactive carbene, which engaged in photoaffinity capture of neighboring proteins. Thirteen proteins were identified. Three of these proteins-PBP1a, PBP5, and MreB-are members of the bacterial divisome. The use of the complementary methodologies of non-canonical amino-acid incorporation, photoaffinity proximity analysis, and fluorescent microscopy confirm a dominant septal location for the RlpA enzyme of P. aeruginosa, as a divisome-associated activity. This accomplishment adds to the emerging recognition of the value of these methodologies for identification of the intracellular localization of bacterial proteins.

Application of 2D-ITC to the Elucidation of the Enzymatic Mechanism of N-Acetylmuramic Acid/N-Acetylglucosamine Kinase (AmgK) from Pseudomonas aeruginosa.

Characterization of the turnover mechanism of bisubstrate enzymes is a tedious task. Molecular tools for studying the enzymatic mechanism are not readily available for all enzymes (e.g., radioactive substrates, substrate-competitive inhibitors, etc.). Wang and Mittermaier recently introduced two-dimensional isothermal titration calorimetry (2D-ITC) for determining the bisubstrate mechanism at high resolution while simultaneously quantifying the kinetic parameters for substrate turnover in a single reporter-free experiment. We demonstrate the utility of 2D-ITC in studying N-acetylmuramic acid/N-acetylglucosamine kinase (AmgK) from Pseudomonas aeruginosa. This enzyme is involved in cytoplasmic cell-wall-recycling events as a step in the peptidoglycan salvage pathway. Furthermore, AmgK phosphorylates N-acetylglucosamine and N-acetylmuramic acid, linking the recycling events to de novo cell-wall synthesis. We document in a 2D-ITC experiment that AmgK follows an ordered-sequential mechanism, where ATP binds first and ADP is released last. We also show that classical enzyme kinetic methods support the results of 2D-ITC and that 2D-ITC could overcome the shortcomings of these classical methodologies. We provide evidence for inhibition of AmgK by the catalytic product ADP, but not by the phosphorylated sugar product. These results provide a full kinetic characterization of the bacterial kinase AmgK. This work highlights 2D-ITC as a versatile tool for the mechanistic evaluation of bisubstrate enzymes, as an alternative for classical methods.

In vitro studies of the protein-interaction network of cell-wall lytic transglycosylase RlpA of Pseudomonas aeruginosa.

The protein networks of cell-wall-biosynthesis assemblies are largely unknown. A key class of enzymes in these assemblies is the lytic transglycosylases (LTs), of which eleven exist in P. aeruginosa. We have undertaken a pulldown strategy in conjunction with mass-spectrometry-based proteomics to identify the putative binding partners for the eleven LTs of P. aeruginosa. A total of 71 putative binding partners were identified for the eleven LTs. A systematic assessment of the binding partners of the rare lipoprotein A (RlpA), one of the pseudomonal LTs, was made. This 37-kDa lipoprotein is involved in bacterial daughter-cell separation by an unknown process. RlpA participates in both the multi-protein and multi-enzyme divisome and elongasome assemblies. We reveal an extensive protein-interaction network for RlpA involving at least 19 proteins. Their kinetic parameters for interaction with RlpA were assessed by microscale thermophoresis, surface-plasmon resonance, and isothermal-titration calorimetry. Notable RlpA binding partners include PBP1b, PBP4, and SltB1. Elucidation of the protein-interaction networks for each of the LTs, and specifically for RlpA, opens opportunities for the study of their roles in the complex protein assemblies intimately involved with the cell wall as a structural edifice critical for bacterial survival.

Integrative structural biology of the penicillin-binding protein-1 from Staphylococcus aureus, an essential component of the divisome machinery.

The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis of PBP1 of Staphylococcus aureus (saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the "penicillin-binding protein and serine/threonine kinase associated" (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domains was determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains of saPBP1 as conformationally mobile and separated from the transpeptidase domain. This conclusion was confirmed by SAXS experiments on the full-length protein in solution. A series of crystallographic complexes with ß-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain in saPBP1 could play a crucial role for in vivo interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.

Exploration of the Structural Space in 4(3H)-Quinazolinone Antibacterials.

We report herein the syntheses of 79 derivatives of the 4(3H)-quinazolinones and their structure-activity relationship (SAR) against methicillin-resistant Staphylococcus aureus (MRSA). Twenty one analogs were further evaluated in in vitro assays. Subsequent investigation of the pharmacokinetic properties singled out compound 73 ((E)-3-(5-carboxy-2-fluorophenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one) for further study. The compound synergized with piperacillin-tazobactam (TZP) both in vitro and in vivo in a clinically relevant mouse model of MRSA infection. The TZP combination lacks activity against MRSA, yet it synergized with compound 73 to kill MRSA in a bactericidal manner. The synergy is rationalized by the ability of the quinazolinones to bind to the allosteric site of penicillin-binding protein (PBP)2a, resulting in opening of the active site, whereby the ß-lactam antibiotic now is enabled to bind to the active site in its mechanism of action. The combination effectively treats MRSA infection, for which many antibiotics (including TZP) have faced clinical obsolescence.

Janus kinase 2 and calcium are required for angiotensin II-dependent activation of steroidogenic acute regulatory protein transcription in H295R human adrenocortical cells.

Angiotensin II- and K+-stimulated aldosterone production in the adrenocortical glomerulosa cells requires induction of the steroidogenic acute regulatory protein (StAR). While both agents activate Ca2+ signaling, the mechanisms leading to aldosterone synthesis are distinct, and the angiotensin II response cannot be mimicked by K+. We previously reported that StAR mRNA levels and promoter-reporter gene activity in transiently transfected H295R human adrenocortical cells were stimulated by angiotensin II but not by K+ treatment. The current study focused on identifying signaling pathways activated by angiotensin II that contribute to StAR transcriptional activation. We show that the angiotensin II-stimulated transcriptional activation of StAR was dependent upon influx of external calcium and requires protein kinase C activation. Furthermore we describe for the first time that the Janus tyrosine kinase family member, JAK2, was activated by angiotensin II treatment of H295R cells. Treatment of the cells with AG490, a selective inhibitor of JAK2, blocked JAK2 activation and StAR reporter gene activity and inhibited steroid production. Taken together these studies describe a novel pathway controlling StAR expression and steroidogenesis in adrenocortical cells.

Alkaline proteinase inhibitor of Pseudomonas aeruginosa: a mutational and molecular dynamics study of the role of N-terminal residues in the inhibition of Pseudomonas alkaline proteinase.

Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five N-terminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the alpha-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2-5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzyme-inhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1' pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant KD by 35-, 180-, and 13-fold, respectively. Mutation at positions 3-5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189-200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.