Cloning and sequence analysis of cDNA for a human homolog of eubacterial ATP-dependent Lon proteases

AbstractOverlapping cDNA clones containing mRNA for a putative Lon protease (LonHS) were isolated from cDNA libraries prepared from human brain poly(A)+ RNA. The determined nucleotide sequence contains a 2814-bp open reading frame with two potential initiation codons (positions 62–64 and 338–340). The 5'-terminal 337-nucleotide fragment of LonHS mRNA is highly enriched with G and C nucleotides and could direct synthesis of the LonHS N-terminal domain. More likely this region promotes initiation of protein synthesis from the second AUG codon in a cap-independent manner. The amino acid sequence initiated at the second AUG codon includes 845 residues, over 30% of which are identical to those of eubacterial Lon proteases. Residues of the ‘A’ and ‘B’ motifs of NTP-binding pattern and a plausible catalytic serine residue are conserved in LonHS. Northern blot analysis revealed LonHS mRNA in lung, duodenum, liver and heart, but not in thymus cells

Saturation Mutagenesis and Molecular Modeling: The Impact of Methionine 182 Substitutions on the Stability of β-Lactamase TEM-1

Serine β-lactamase TEM-1 is the first β-lactamase discovered and is still common in Gram-negative pathogens resistant to β-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a “global suppressor”, has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of β-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the β-lactamase to be reactivated

Drug Repurposing of the Unithiol: Inhibition of Metallo-β-Lactamases for the Treatment of Carbapenem-Resistant Gram-Negative Bacterial Infections

The increasing antibiotic resistance is a clinical problem worldwide. Numerous Gram-negative bacteria have already become resistant to the most widely used class of antibacterial drugs, β-lactams. One of the main mechanisms is inactivation of β-lactam antibiotics by bacterial β-lactamases. Appearance and spread of these enzymes represent a continuous challenge for the clinical treatment of infections and for the design of new antibiotics and inhibitors. Drug repurposing is a prospective approach for finding new targets for drugs already approved for use. We describe here the inhibitory potency of known detoxifying antidote 2,3-dimercaptopropane-1-sulfonate (unithiol) against metallo-β-lactamases. Unithiol acts as a competitive inhibitor of meropenem hydrolysis by recombinant metallo-β-lactamase NDM-1 with the KI of 16.7 µM. It is an order of magnitude lower than the KI for l-captopril, the inhibitor of angiotensin-converting enzyme approved as a drug for the treatment of hypertension. Phenotypic methods demonstrate that the unithiol inhibits natural metallo-β-lactamases NDM-1 and VIM-2 produced by carbapenem-resistant K. pneumoniae and P. aeruginosa bacterial strains. The 3D full atom structures of unithiol complexes with NDM-1 and VIM-2 are obtained using QM/MM modeling. The thiol group is located between zinc cations of the active site occupying the same place as the catalytic hydroxide anion in the enzyme–substrate complex. The sulfate group forms both a coordination bond with a zinc cation and hydrogen bonds with the positively charged residue, lysine or arginine, responsible for proper orientation of antibiotics upon binding to the active site prior to hydrolysis. Thus, we demonstrate both experimentally and theoretically that the unithiol is a prospective competitive inhibitor of metallo-β-lactamases and it can be utilized in complex therapy together with the known β-lactam antibiotics

Crystal structures of the molecular class A β-lactamase TEM-171 and its complexes with tazobactam

The resistance of bacteria to β-lactam antibiotics is primarily caused by the production of β-lactamases. Here, novel crystal structures of the native β-lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical β-lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder. The tazobactam intermediate was covalently bound to the catalytic Ser70 in the trans-enamine configuration. While the conformation of tazobactam in the first complex resembled that in published β-lactamase–tazobactam structures, in the second complex, which was obtained after longer soaking of the native crystals in the inhibitor solution, a new and previously unreported tazobactam conformation was observed. It is proposed that the two complexes correspond to different stages along the deacylation path of the acyl-enzyme intermediate. The results provide a novel structural basis for the rational design of new β-lactamase inhibitors

The 6^{6}H states studied in the d(8He,α)d(^8\text{He},\alpha) reaction and evidence of extremely correlated character of the 5^{5}H ground state

The extremely neutron-rich system $^{6}$H was studied in the direct $^2\text{H}(^8\text{He},{^4\text{He}})^{6}$H transfer reaction with a 26 $A$MeV secondary $^{8}$He beam. The measured missing mass spectrum shows a resonant state in $^{6}$H at $6.8(3)$ MeV relative to the $^3$H+$3n$ threshold. There is also some evidence of a resonant state at $4.5(3)$ MeV which is a realistic candidate for the $^{6}$H ground state (g.s.). The population cross section of the presumably $p$-wave states in the energy range from 4 to 8 MeV is $d\sigma/d\Omega_{\text{c.m.}} \sim 190$$\mu$b/sr in the angular range $5^{\circ}<\theta_{\text{c.m.}}<16^{\circ}$. The obtained missing mass spectrum is free of the $^{6}$H events below 3.5 MeV ($d\sigma/d\Omega_{\text{c.m.}} \lesssim 3$$\mu$b/sr in the angular range $5^{\circ}<\theta_{\text{c.m.}}<20^{\circ}$), which indicates that the value of 4.5 MeV is the lower limit of the possible $^{6}$H g.s. location. The obtained results confirm that the decay mechanism of the $^{7}$H g.s. (located at 2.2 MeV above the $^{3}$H+$4n$ threshold) is the ``true'' (or simultaneous) $4n$ emission. The resonance energy profiles and the momentum distributions of the sequential $^{6}$H \,\rightarrow \, ^5H(g.s.)+n\, \rightarrow \, ^3H+$3n$ decay fragments were analyzed by the theoretically-updated direct four-body-decay and sequential-emission mechanisms. The measured momentum distributions of the $^{3}$H fragments in the $^{6}$H rest frame indicate a very strong ``dineutron-type'' correlations in the $^{5}$H ground state decay