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Smith, Clyde A.

Publications and source records attributed to Smith, Clyde A..

10 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Decarboxylation of the Catalytic Lysine Residue by the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the OXA-58 Carbapenemase

Antibiotic resistance in bacteria is a major global health concern. The wide spread of carbapenemases, bacterial enzymes that degrade the last-resort carbapenem antibiotics, is responsible for multidrug resistance in bacterial pathogens and has further significantly exacerbated this problem. Acinetobacter baumannii is one of the leading nosocomial pathogens due to the acquisition and wide dissemination of carbapenem-hydrolyzing class D β-lactamases, which have dramatically diminished available therapeutic options. Thus, new antibiotics that are active against multidrug-resistantA. baumannii and carbapenemase inhibitors are urgently needed. Here we report characterization of the interaction of the C5α-methyl-substituted carbapenem NA-1-157 with one of the clinically important class D carbapenemases, OXA-58. Antibiotic susceptibility testing shows that the compound is more potent than commercial carbapenems against OXA-58-producingA. baumannii, with a clinically sensitive MIC value of 1 μg/mL. Kinetic studies demonstrate that NA-1-157 is a very poor substrate of the enzyme due mainly to a significantly reduced deacylation rate. Mass spectrometry analysis shows that inhibition of OXA-58 by NA-1-157 proceeds through both the classical acyl-enzyme intermediate and a reversible covalent species. Time-resolved X-ray crystallographic studies reveal that upon acylation of the enzyme, the compound causes progressive decarboxylation of the catalytic lysine residue, thus severely impairing deacylation. Overall, this study demonstrates that the carbapenem NA-1-157 is highly resistant to degradation by the OXA-58 carbapenemase.

Acinetobactor↗
DOE OSTI2024

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 (k 3 = (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. Furthermore, these data demonstrate that NA-1-157 is a potent irreversible inhibitor of the GES-5 carbapenemase.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

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

Pea (legume plant defense) phytoalexin pterocarpans (–)-maackiain and (+)-pisatin have opposite C6a and C11a configurations, but how this occurs biosynthetically is unknown. Herein, pea dirigent-protein (DP) PsPTS2, which generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), was investigated as regards stereoselectivity towards four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers. Each DMDI stereoisomer configuration was determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF, this being 20-fold faster than with the trans-(3R,4S)-isomer. The 4R-configured substrate’s near ß-axial OH orientation significantly enhanced its leaving group abilities in creating intermediate A-ring mono-quinone methide (QM), whereas the 4S-isomer’s 4-OH group α-equatorial orientation was a poorer leaving group. Substrate docking simulations also indicated that the 4R-configured ß-axial OH was closest to the Asp catalytic center, whereas the 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 the same C3/C4 stereochemistry as (+)-pisatin. PsPTS2, assayed with cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and its C3/C4 stereoisomers, gave 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF), with the same substrate stereoselectivity. DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and the tree legume Bolusanthus speciosus, as DMIF is found in both. PsPTS1 preferentially utilized cis-(3R,4R)-DMDI to give (–)-maackiain, this being 2200-fold more efficient than with cis-(3R,4R)-DMI. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, again reflecting the better 4R configured OH leaving group. Pea PsPTS2 and PsPTS1 provisionally provide a facile DP-engendered means to enable differing C6a and C11a configurations in (+)-pisatin and (–)-maackiain. Both DP transformations are considered to occur via DP-engendered generation of an identical mono-QM bound intermediate, which with PsPTS2 either re-aromatizes to give DMDIF or with PsPTS1 undergoes intramolecular cyclization to afford (–)-maackiain. Substrate docking simulations using PsPTS2 (2.5 Å resolution) and PsPTS1 (1.5 Å resolution) plausibly indicate that cis-(3R,4R)-DMDI binds in the anti-configuration in the PsPTS2 active site to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

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 β-lactam antibiotics by producing β-lactamases, enzymes which destroy these valuable agents. Historically, the carbapenem family of b-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 β-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-β-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. 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 β-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

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

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.

59 BASIC BIOLOGICAL SCIENCES↗