Restricted Rotational Flexibility of the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the GES-5 Carbapenemase.

Carbapenem antibiotics are used as a last-resort treatment for infections caused by multidrug-resistant bacteria. The wide spread of carbapenemases in Gram-negative bacteria has severely compromised the utility of these drugs and represents a serious public health threat. To combat carbapenemase-mediated resistance, new antimicrobials and inhibitors of these enzymes are urgently needed. Here, we describe the interaction of the atypically C5α-methyl-substituted carbapenem, NA-1-157, with the GES-5 carbapenemase. MICs of this compound against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii producing the enzyme were reduced 4-16-fold when compared to MICs of the commercial carbapenems, reaching clinically sensitive breakpoints. When NA-1-157 was combined with meropenem, a strong synergistic effect was observed. Kinetic and ESI-LC/MS studies demonstrated that NA-1-157 is a potent inhibitor of GES-5, with a high inactivation efficiency of (2.9 ± 0.9) × 105 M-1 s-1. Acylation of GES-5 by NA-1-157 was biphasic, with the fast phase completing within seconds, and the slow phase taking several hours and likely proceeding through a reversible tetrahedral intermediate. Deacylation was extremely slow (k3 = (2.4 ± 0.3) × 10-7 s-1), resulting in a residence time of 48 ± 6 days. MD simulation of the GES-5-meropenem and GES-5-NA-1-157 acyl-enzyme complexes revealed that the C5α-methyl group in NA-1-157 sterically restricts rotation of the 6α-hydroxyethyl group preventing ingress of the deacylating water into the vicinity of the scissile bond of the acyl-enzyme intermediate. These data demonstrate that NA-1-157 is a potent irreversible inhibitor of the GES-5 carbapenemase.

Novel sterol binding domains in bacteria.

Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.

Dirigent isoflavene-forming PsPTS2: 3D structure, stereochemical, and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea.

Pea phytoalexins (-)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity toward four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF 20-fold faster than the trans-(3R,4S)-isomer. The 4R-configured substrate's near ß-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S-isomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured ß-axial OH was closest to Asp51, whereas 4S-isomer's α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and C3/C4 stereoisomers to give 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF). DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and tree legume Bolusanthus speciosus, as DMIF occurs in both species. PsPTS1 utilized cis-(3R,4R)-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3R,4R)-DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (-)-maackiain, via identical DP-engendered mono-QM bound intermediate generation, which PsPTS2 either re-aromatizes to give DMDIF or PsPTS1 intramolecularly cyclizes to afford (-)-maackiain. Substrate docking simulations using PsPTS2 and PsPTS1 indicate cis-(3R,4R)-DMDI binds in the anti-configuration in PsPTS2 to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain.

The C5α-Methyl-Substituted Carbapenem NA-1-157 Exhibits Potent Activity against Klebsiella spp. Isolates Producing OXA-48-Type Carbapenemases.

The wide spread of carbapenem-hydrolyzing ß-lactamases in Gram-negative bacteria has diminished the utility of the last-resort carbapenem antibiotics, significantly narrowing the available therapeutic options. In the Enterobacteriaceae family, which includes many important clinical pathogens such as Klebsiella pneumoniae and Escherichia coli, production of class D ß-lactamases from the OXA-48-type family constitutes the major mechanism of resistance to carbapenems. To address the public health threat posed by these enzymes, novel, effective therapeutics are urgently needed. Here, we report evaluation of a novel, C5α-methyl-substituted carbapenem, NA-1-157, and show that its MICs against bacteria producing OXA-48-type enzymes were reduced by 4- to 32-fold when compared to meropenem. When combined with commercial carbapenems, the potency of NA-1-157 was further enhanced, resulting in target potentiation concentrations ranging from 0.125 to 2 µg/mL. Kinetic studies demonstrated that the compound is poorly hydrolyzed by OXA-48, with a catalytic efficiency 30- to 50-fold lower than those of imipenem and meropenem. Acylation of OXA-48 by NA-1-157 was severely impaired, with a rate 10,000- to 36,000-fold slower when compared to the commercial carbapenems. Docking, molecular dynamics, and structural studies demonstrated that the presence of the C5α-methyl group in NA-1-157 creates steric clashes within the active site, leading to differences in the position and the hydrogen-bonding pattern of the compound, which are incompatible with efficient acylation. This study demonstrates that NA-1-157 is a promising novel carbapenem for treatment of infections caused by OXA-48-producing bacterial pathogens.

Dirigent protein subfamily function and structure in terrestrial plant phenol metabolism.

Aquatic plant transition to land, and subsequent terrestrial plant species diversification, was accompanied by the emergence and massive elaboration of plant phenol chemo-diversity. Concomitantly, dirigent protein (DP) and dirigent-like protein subfamilies, derived from large multigene families, emerged and became extensively diversified. DP biochemical functions as gateway entry points into new and diverse plant phenol skeletal types then markedly expanded. DPs have at least eight non-uniformly distributed subfamilies, with different DP subfamily members of known biochemical/physiological function now implicated as gateway entries to lignan, lignin, aromatic diterpenoid, pterocarpan and isoflavene pathways. While some other DP subfamily members have jacalin domains, both these and indeed the majority of DPs throughout the plant kingdom await discovery of their biochemical roles. Methods and approaches were developed to discover DP biochemical function as gateway entry points to distinct plant phenol skeletal types in land plants. Various DP 3D X-ray structural determinations enabled structure-based comparative sequence analysis and modeling to understand similarities and differences among the different DP subfamilies. We consider that the core DP ß-barrel fold and associated characteristics are likely common to all DPs, with several residues conserved and nearly invariant. There is also considerable variation in residue composition and topography of the putative substrate binding pockets, as well as substantial differences in several loops, such as the ß1-ß2 loop. All DPs likely bind and stabilize quinone methide intermediates, while guiding distinctive regio- and/or stereo-chemical entry into Nature's chemo-diverse land plant phenol metabolic classes.

Structural characterization of a soil viral auxiliary metabolic gene product - a functional chitosanase.

Metagenomics is unearthing the previously hidden world of soil viruses. Many soil viral sequences in metagenomes contain putative auxiliary metabolic genes (AMGs) that are not associated with viral replication. Here, we establish that AMGs on soil viruses actually produce functional, active proteins. We focus on AMGs that potentially encode chitosanase enzymes that metabolize chitin - a common carbon polymer. We express and functionally screen several chitosanase genes identified from environmental metagenomes. One expressed protein showing endo-chitosanase activity (V-Csn) is crystalized and structurally characterized at ultra-high resolution, thus representing the structure of a soil viral AMG product. This structure provides details about the active site, and together with structure models determined using AlphaFold, facilitates understanding of substrate specificity and enzyme mechanism. Our findings support the hypothesis that soil viruses contribute auxiliary functions to their hosts.

The l,d-Transpeptidase LdtAb from Acinetobacter baumannii Is Poorly Inhibited by Carbapenems and Has a Unique Structural Architecture.

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3-3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of ß-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of LdtAb from the clinically important pathogen, Acinetobacter baumannii. We show that A. baumannii survives inactivation of LdtAb alone or in combination with PBP1b or PBP2, while simultaneous inactivation of LdtAb and PBP1a is lethal. Minimal inhibitory concentrations (MICs) of all 13 ß-lactam antibiotics tested decreased 2- to 8-fold for the LdtAb deletion mutant, while further decreases were seen for both double mutants, with the largest, synergistic effect observed for the LdtAb + PBP2 deletion mutant. Mass spectrometry experiments showed that LdtAb forms complexes in vitro only with carbapenems. However, the acylation rate of these antibiotics is very slow, with the reaction taking longer than four hours to complete. Our X-ray crystallographic studies revealed that LdtAb has a unique structural architecture and is the only known LDT to have two different peptidoglycan-binding domains.

Esomeprazole covalently interacts with the cardiovascular enzyme dimethylarginine dimethylaminohydrolase: Insights into the cardiovascular risk of proton pump inhibitors.

BACKGROUND: Proton pump inhibitors (PPIs) are widely prescribed drugs for the treatment of gastroesophageal reflux disease (GERD). Several meta-analysis studies have reported associations between prolonged use of PPIs and major adverse cardiovascular events. However, interaction of PPIs with biological molecules involved in cardiovascular health is incompletely characterized. Dimethylarginine dimethylaminohydrolase (DDAH) is a cardiovascular enzyme expressed in cardiomyocytes, and other somatic cell types in one of two isotypes (DDAH1 and DDAH2) to metabolize asymmetric dimethylarginine (ADMA); a cardiovascular risk factor and competitive inhibitor of nitric oxide synthases (NOSs). METHODS: We performed high throughput drug screening of over 130,000 small molecules to discover human DDAH1 inhibitors and found that PPIs directly inhibit DDAH1. We expressed and purified the enzyme for structural and mass spectrometry proteomics studies to understand how a prototype PPI, esomeprazole, interacts with DDAH1. We also performed molecular docking studies to model the interaction of DDAH1 with esomeprazole. X-ray crystallography was used to determine the structure of DDAH1 alone and bound to esomeprazole at resolutions ranging from 1.6 to 2.9 Å. RESULTS: Analysis of the enzyme active site shows that esomeprazole interacts with the active site cysteine (Cys273) of DDAH1. The structural studies were corroborated by mass spectrometry which indicated that cysteine was targeted by esomeprazole to inactivate DDAH1. CONCLUSIONS: The inhibition of this important cardiovascular enzyme by a PPI may help explain the reported association of PPI use and increased cardiovascular risk in patients and the general population. GENERAL SIGNIFICANCE: Our study calls for pharmacovigilance studies to monitor adverse cardiovascular events in chronic PPI users.

C6 Hydroxymethyl-Substituted Carbapenem MA-1-206 Inhibits the Major Acinetobacter baumannii Carbapenemase OXA-23 by Impeding Deacylation.

Acinetobacter baumannii has become a major nosocomial pathogen, as it is often multidrug-resistant, which results in infections characterized by high mortality rates. The bacterium achieves high levels of resistance to ß-lactam antibiotics by producing ß-lactamases, enzymes which destroy these valuable agents. Historically, the carbapenem family of ß-lactam antibiotics have been the drugs of choice for treating A. baumannii infections. However, their effectiveness has been significantly diminished due to the pathogen's production of carbapenem-hydrolyzing class D ß-lactamases (CHDLs); thus, new antibiotics and inhibitors of these enzymes are urgently needed. Here, we describe a new carbapenem antibiotic, MA-1-206, in which the canonical C6 hydroxyethyl group has been replaced with hydroxymethyl. The antimicrobial susceptibility studies presented here demonstrated that this compound is more potent than meropenem and imipenem against A. baumannii producing OXA-23, the most prevalent CHDL of this pathogen, and also against strains producing the CHDL OXA-24/40 and the class B metallo-ß-lactamase VIM-2. Our kinetic and mass spectrometry studies revealed that this drug is a reversible inhibitor of OXA-23, where inhibition takes place through a branched pathway. X-ray crystallographic studies, molecular docking, and molecular dynamics simulations of the OXA-23-MA-1-206 complex show that the C6 hydroxymethyl group forms a hydrogen bond with the carboxylated catalytic lysine of OXA-23, effectively preventing deacylation. These results provide a promising strategy for designing a new generation of CHDL-resistant carbapenems to restore their efficacy against deadly A. baumannii infections. IMPORTANCE Carbapenem antibiotics are the drugs of choice for treatment of deadly infections caused by Gram-negative bacteria. However, their efficacy is severely compromised by the wide spread of carbapenem-hydrolyzing class D ß-lactamases (CHDLs). The importance of this research is the discovery that substitution of the canonical hydroxyethyl group of carbapenems by a hydroxymethyl significantly enhances stability against inactivation by the major CHDL of Acinetobacter baumannii, OXA-23. These results provide a novel strategy for designing next-generation, carbapenemase-stable carbapenems to fight multidrug-resistant infections caused by Gram-negative pathogens.

Making sense of SFX data: standards for data and structure validation for a non-standard experiment that has come of age.

SFX diffraction data collection at XFELs is becoming more accessible. To extract the most useful biological information from these non-standard experiments, standards for SFX data analysis and structure validation must be redefined.

Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

In Crystallo Time-Resolved Interaction of the Clostridioides difficile CDD-1 enzyme with Avibactam Provides New Insights into the Catalytic Mechanism of Class D ß-lactamases.

Class D ß-lactamases have risen to notoriety due to their wide spread in bacterial pathogens, propensity to inactivate clinically important ß-lactam antibiotics, and ability to withstand inhibition by the majority of classical ß-lactamase inhibitors. Understanding the catalytic mechanism of these enzymes is thus vitally important for the development of novel antibiotics and inhibitors active against infections caused by antibiotic-resistant bacteria. Here we report an in crystallo time-resolved study of the interaction of the class D ß-lactamase CDD-1 from Clostridioides difficile with the diazobicyclooctane inhibitor, avibactam. We show that the catalytic carboxylated lysine, a residue that is essential for both acylation and deacylation of ß-lactams, is sequestered within an internal sealed pocket of the enzyme. Time-resolved snapshots generated in this study allowed us to observe decarboxylation of the lysine and movement of CO2 and water molecules through a transient channel formed between the lysine pocket and the substrate binding site facilitated by rotation of the side chain of a conserved leucine residue. These studies provide novel insights on avibactam binding to CDD-1 and into the catalytic mechanism of class D ß-lactamases in general.

Inhibition of the Clostridioides difficile Class D ß-Lactamase CDD-1 by Avibactam.

Avibactam is a potent diazobicyclooctane inhibitor of class A and C ß-lactamases. The inhibitor also exhibits variable activity against some class D enzymes from Gram-negative bacteria; however, its interaction with recently discovered class D ß-lactamases from Gram-positive bacteria has not been studied. Here, we describe microbiological, kinetic, and mass spectrometry studies of the interaction of avibactam with CDD-1, a class D ß-lactamase from the clinically important pathogen Clostridioides difficile, and show that avibactam is a potent irreversible mechanism-based inhibitor of the enzyme. X-ray crystallographic studies at three time-points demonstrate the rapid formation of a stable CDD-1-avibactam acyl-enzyme complex and highlight differences in the anchoring of the inhibitor by class D enzymes from Gram-positive and Gram-negative bacteria.

Crystal structures of glutathione- and inhibitor-bound human GGT1: critical interactions within the cysteinylglycine binding site.

Overexpression of γ-glutamyl transpeptidase (GGT1) has been implicated in an array of human diseases including asthma, reperfusion injury, and cancer. Inhibitors are needed for therapy, but development of potent, specific inhibitors of GGT1 has been hampered by a lack of structural information regarding substrate binding and cleavage. To enhance our understanding of the molecular mechanism of substrate cleavage, we have solved the crystal structures of human GGT1 (hGGT1) with glutathione (a substrate) and a phosphate-glutathione analog (an irreversible inhibitor) bound in the active site. These are the first structures of any eukaryotic GGT with the cysteinylglycine region of the substrate-binding site occupied. These structures and the structure of apo-hGGT reveal movement of amino acid residues within the active site as the substrate binds. Asn-401 and Thr-381 each form hydrogen bonds with two atoms of GSH spanning the γ-glutamyl bond. Three different atoms of hGGT1 interact with the carboxyl oxygen of the cysteine of GSH. Interactions between the enzyme and substrate change as the substrate moves deeper into the active site cleft. The substrate reorients and a new hydrogen bond is formed between the substrate and the oxyanion hole. Thr-381 is locked into a single conformation as an acyl bond forms between the substrate and the enzyme. These data provide insight on a molecular level into the substrate specificity of hGGT1 and provide an explanation for seemingly disparate observations regarding the enzymatic activity of hGGT1 mutants. This knowledge will aid in the design of clinically useful hGGT1 inhibitors.

Fragment Binding to the Nsp3 Macrodomain of SARS-CoV-2 Identified Through Crystallographic Screening and Computational Docking.

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

A surface loop modulates activity of the Bacillus class D ß-lactamases.

The expression of ß-lactamases is a major mechanism of bacterial resistance to the ß-lactam antibiotics. Four molecular classes of ß-lactamases have been described (A, B, C and D), however until recently the class D enzymes were thought to exist only in Gram-negative bacteria. In the last few years, class D enzymes have been discovered in several species of Gram-positive microorganisms, such as Bacillus and Clostridia, and an investigation of their kinetic and structural properties has begun in earnest. Interestingly, it was observed that some species of Bacillus produce two distinct class D ß-lactamases, one highly active and the other with only basal catalytic activity. Analysis of amino acid sequences of active (BPU-1 from Bacillus pumilus) and inactive (BSU-2 from Bacillus subtilis and BAT-2 from Bacillus atrophaeus) enzymes suggests that presence of three additional amino acid residues in one of the surface loops of inefficient ß-lactamases may be responsible for their severely diminished activity. Our structural and docking studies show that the elongated loop of these enzymes severely restricts binding of substrates. Deletion of the three residues from the loops of BSU-2 and BAT-2 ß-lactamases relieves the steric hindrance and results in a significant increase in the catalytic activity of the enzymes. These data show that this surface loop plays an important role in modulation of the catalytic activity of Bacillus class D ß-lactamases.

Pterocarpan synthase (PTS) structures suggest a common quinone methide-stabilizing function in dirigent proteins and proteins with dirigent-like domains.

The biochemical activities of dirigent proteins (DPs) give rise to distinct complex classes of plant phenolics. DPs apparently began to emerge during the aquatic-to-land transition, with phylogenetic analyses revealing the presence of numerous DP subfamilies in the plant kingdom. The vast majority (>95%) of DPs in these large multigene families still await discovery of their biochemical functions. Here, we elucidated the 3D structures of two pterocarpan-forming proteins with dirigent-like domains. Both proteins stereospecifically convert distinct diastereomeric chiral isoflavonoid precursors to the chiral pterocarpans, (-)- and (+)-medicarpin, respectively. Their 3D structures enabled comparisons with stereoselective lignan- and aromatic terpenoid-forming DP orthologs. Each protein provides entry into diverse plant natural products classes, and our experiments suggest a common biochemical mechanism in binding and stabilizing distinct plant phenol-derived mono- and bis-quinone methide intermediates during different C-C and C-O bond-forming processes. These observations provide key insights into both their appearance and functional diversification of DPs during land plant evolution/adaptation. The proposed biochemical mechanisms based on our findings provide important clues to how additional physiological roles for DPs and proteins harboring dirigent-like domains can now be rationally and systematically identified.

Lucina pectinata oxyhemoglobin (II-III) heterodimer pH susceptibility.

Lucina pectinata live in high concentrations of hydrogen sulfide (H2S) and contains one hemoglobin, Hemoglobin I (HbI), transporting H2S and two hemoglobins, Hemoglobin II (HbII) and Hemoglobin (HbIII), transferring dioxygen to symbionts. HbII and HbIII contain B10 tyrosine (Tyr) and E7 glutamine (Gln) in the heme pocket generating an efficient hydrogen bonding network with the (HbII-HbIII)-O2 species, leading to very low ligand dissociation rates. The results indicate that the oxy-hemeprotein is susceptible to pH from 4 to 9, at acidic conditions, and as a function of the potassium ferricyanide concentration, 100% of the met-aquo derivative is produced. Without a strong oxidant, pH 5 generates a small concentration of the met-aquo complex. The process is accelerated by the presence of salts, as indicated by the crystallization structures and UV-Vis spectra. The results suggest that acidic pH generates conformational changes associated with B10 and E7 heme pocket amino acids, weakening the (HbII-HbIII)-O2 hydrogen bond network. The observation is supported by X-ray crystallography, since at pH 4 and 5, the heme-Fe tends to oxidize, while at pH 7, the oxy-heterodimer is present. Conformational changes also are observed at higher pH by the presence of a 605 nm transition associated with the iron heme-Tyr interaction. Therefore, pH is one crucial factor regulating the (HbII-HbIII)-O2 complex hydrogen-bonding network. Thus, it can be proposed that the hydrogen bonding adjustments between the heme bound O2 and the Tyr and Gln amino acids contribute to oxygen dissociation from the (HbII-HbIII)-O2 system.

Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2''-phosphotransferases.

Aminoglycoside phosphotransferases (APHs) are one of three families of aminoglycoside-modifying enzymes that confer high-level resistance to the aminoglycoside antibiotics via enzymatic modification. This has now rendered many clinically important drugs almost obsolete. The APHs specifically phosphorylate hydroxyl groups on the aminoglycosides using a nucleotide triphosphate as the phosphate donor. The APH(2'') family comprises four distinct members, isolated primarily from Enterococcus sp., which vary in their substrate specificities and also in their preference for the phosphate donor (ATP or GTP). The structure of the ternary complex of APH(2'')-IIIa with GDP and kanamycin was solved at 1.34 Šresolution and was compared with substrate-bound structures of APH(2'')-Ia, APH(2'')-IIa and APH(2'')-IVa. In contrast to the case for APH(2'')-Ia, where it was proposed that the enzyme-mediated hydrolysis of GTP is regulated by conformational changes in its N-terminal domain upon GTP binding, APH(2'')-IIa, APH(2'')-IIIa and APH(2'')-IVa show no such regulatory mechanism, primarily owing to structural differences in the N-terminal domains of these enzymes.

The crystal structures of CDD-1, the intrinsic class D ß-lactamase from the pathogenic Gram-positive bacterium Clostridioides difficile, and its complex with cefotaxime.

Class D ß-lactamases, enzymes that degrade ß-lactam antibiotics and are widely spread in Gram-negative bacteria, were for a long time not known in Gram-positive organisms. Recently, these enzymes were identified in various non-pathogenic Bacillus species and subsequently in Clostridioides difficile, a major clinical pathogen associated with high morbidity and mortality rates. Comparison of the BPU-1 enzyme from Bacillus pumilus with the CDD-1 and CDD-2 enzymes from C. difficile demonstrated that the latter enzymes have broadened their substrate profile to efficiently hydrolyze the expanded-spectrum methoxyimino cephalosporins, cefotaxime and ceftriaxone. These two antibiotics are major contributors to the development of C. difficile infection, as they suppress sensitive bacterial microflora in the gut but fail to kill the pathogen which is highly resistant to these drugs. To gain insight into the structural features that contribute to the expansion of the substrate profile of CDD enzymes compared to BPU-1, we solved the crystal structures of CDD-1 and its complex with cefotaxime. Comparison of CDD-1 structures with those of class D enzymes from Gram-negative bacteria showed that in the cefotaxime-CDD-1 complex, the antibiotic is bound in a substantially different mode due to structural differences in the enzymes' active sites. We also found that CDD-1 has a uniquely long Ω-loop when compared to all other class D ß-lactamases. This Ω-loop extension allows it to engage in hydrogen bonding with the acylated cefotaxime, thus providing additional stabilizing interactions with the substrate which could be responsible for the high catalytic activity of the enzyme for expanded-spectrum cephalosporins.