ABSTRACT
In the chapters dealing with enzyme reactions, the authors of all Biochemistry textbooks and of even more specialized texts consider that the characteristic parameters (kcat and Km) must be determined under initial or steady-state rate conditions. This implies the transformation of a very limited proportion of substrate (at most 10-20%) or a continuous recording of the product or substrate concentration vs. time. Both options can present practical difficulties. Is it possible to get around these very stringent conditions? Here we show that in the most favourable cases up to 70% of the substrate can be converted resulting in systematic errors on the parameters (that can easily be taken account of) if the simple Henri-Michaelis-Menten equation is utilised. Alternatively, the integrated form of the same equation directly yields excellent estimates of the same parameters. Our observations should greatly facilitate the task of researchers who study systems in which measurements of the reaction progress are painstaking or when substrate concentrations close to the detection limit must be used. The general conclusion is that it is not always absolutely necessary to determine initial or steady-state rates to obtain reliable estimations of the enzyme kinetic parameters..
Subject(s)
Physics , Research Personnel , HumansABSTRACT
Fungi are of great importance in biotechnology, for example in the production of enzymes and metabolites. The main goal of this study was to obtain a high-coverage draft of the Stachybotrys microspora genome and to annotate and analyze the genome sequence data. The rare fungus S. microspora N1 strain is distinguished by its ability to grow in an alkaline halophilic environment and to efficiently secrete cellulolytic enzymes. Here we report the draft genome sequence composed of 3715 contigs, a genome size of 35 343 854 bp, with a GC content of 53.31% and a coverage around 20.5×. The identification of cellulolytic genes and of their corresponding functions was carried out through analysis and annotation of the whole genome sequence. Forty-six cellulases were identified using the fungicompanion bioinformatic tool. Interestingly, an S. microspora endoglucanase selected from those with a low isoelectric point was predicted to have a halophilic profile and share significant homology with a well-known bacterial halophilic cellulase. These results confirm previous biochemical studies revealing a halophilic character, which is a very rare feature among fungal cellulases. All these properties suggest that cellulases of S. microspora may have potential for use in the biofuel, textile, and detergent industries.
Subject(s)
Cellulase , Cellulases , Stachybotrys , Cellulase/genetics , Cellulase/chemistry , Cellulase/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Cellulases/genetics , Cellulases/metabolism , Stachybotrys/genetics , Stachybotrys/metabolismABSTRACT
Monocyclic ß-lactams revive the research field on antibiotics, which are threatened by the emergence of resistant bacteria. A six-step synthetic route was developed, providing easy access to new 3-amino-1-carboxymethyl-4-phenyl-ß-lactams, of which the penicillin-binding protein (PBP) inhibitory potency was demonstrated biochemically.
Subject(s)
Amino Acids/pharmacology , Anti-Bacterial Agents/pharmacology , Dipeptides/pharmacology , Imines/pharmacology , Lactams/pharmacology , Penicillin-Binding Proteins/antagonists & inhibitors , Amino Acids/chemistry , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Dipeptides/chemical synthesis , Dipeptides/chemistry , Imines/chemistry , Lactams/chemical synthesis , Lactams/chemistry , Molecular Structure , Penicillin-Binding Proteins/metabolismABSTRACT
Innovative monocyclic ß-lactam entities create opportunities in the battle against resistant bacteria because of their PBP acylation potential, intrinsically high ß-lactamase stability and compact scaffold. α-Benzylidene-substituted 3-amino-1-carboxymethyl-ß-lactams were recently shown to be potent PBP inhibitors and constitute eligible anchor points for synthetic elaboration of the chemical space around the central ß-lactam ring. The present study discloses a 12-step synthesis of ten α-arylmethylidenecarboxylates using a microwave-assisted Wittig olefination as the crucial reaction step. The library was designed aiming at enhanced ß-lactam electrophilicity and extended electron flow after enzymatic attack. Additionally, increased ß-lactamase stability and intermolecular target interaction were envisioned by tackling both the substitution pattern of the aromatic ring and the ß-lactam C4-position. The significance of α-unsaturation was validated and the R39/PBP3 inhibitory potency shown to be augmented the most through decoration of the aromatic ring with electron-withdrawing groups. Furthermore, ring cleavage by representative ß-lactamases was ruled out, providing new insights in the SAR landscape of monocyclic ß-lactams as eligible PBP or ß-lactamase inhibitors.
ABSTRACT
Transcriptomes consist of several classes of RNA that have wide-ranging but often poorly described functions and the deregulation of which leads to numerous diseases. Engineering of functionalized RNA-binding proteins (RBPs) could therefore have many applications. Our previous studies suggested that the RanBP2-type Zinc Finger (ZF) domain is a suitable scaffold to investigate the design of single-stranded RBPs. In the present work, we have analyzed the natural sequence specificity of various members of the RanBP2-type ZF family and characterized the interaction with their target RNA. Surprisingly, our data showed that natural RanBP2-type ZFs with different RNA-binding residues exhibit a similar sequence specificity and therefore no simple recognition code can be established. Despite this finding, different discriminative abilities were observed within the family. In addition, in order to target a long RNA sequence and therefore gain in specificity, we generated a 6-ZF array by combining ZFs from the RanBP2-type family but also from different families, in an effort to achieve a wider target sequence repertoire. We showed that this chimeric protein recognizes its target sequence (20 nucleotides), both in vitro and in living cells. Altogether, our results indicate that the use of ZFs in RBP design remains attractive even though engineering of specificity changes is challenging.
Subject(s)
RNA-Binding Proteins/genetics , SELEX Aptamer Technique/methods , Base Sequence , Binding Sites , Drug Design , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Engineering , RNA/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Structure-Activity Relationship , Zinc FingersABSTRACT
As a complement to the renowned bicyclic ß-lactam antibiotics, monocyclic analogues provide a breath of fresh air in the battle against resistant bacteria. In that framework, the present study discloses the in silico design and unprecedented ten-step synthesis of eleven nocardicin-like enantiomerically pure 2-{3-[2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido]-2-oxoazetidin-1-yl}acetic acids starting from serine as a readily accessible precursor. The capability of this novel class of monocyclic 3-amino-ß-lactams to inhibit penicillin-binding proteins (PBPs) of various (resistant) bacteria was assessed, revealing the potential of α-benzylidenecarboxylates as interesting leads in the pursuit of novel PBP inhibitors. No deactivation by representative enzymes belonging to the four ß-lactamase classes was observed, while weak inhibition of class C ß-lactamase P99 was demonstrated.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Enterococcus faecium/drug effects , Escherichia coli/drug effects , Penicillin-Binding Proteins/antagonists & inhibitors , beta-Lactams/chemistry , beta-Lactams/pharmacology , Amination , Anti-Bacterial Agents/chemical synthesis , Bacterial Infections/drug therapy , Computer Simulation , Computer-Aided Design , Drug Design , Drug Resistance, Bacterial/drug effects , Enterococcus faecium/metabolism , Escherichia coli/metabolism , Escherichia coli Infections/drug therapy , Gram-Positive Bacterial Infections/drug therapy , Humans , Molecular Docking Simulation , Penicillin-Binding Proteins/metabolism , beta-Lactams/chemical synthesisABSTRACT
Phormidesmis priestleyi ULC007 is an Antarctic freshwater cyanobacterium. Its draft genome is 5,684,389 bp long. It contains a total of 5,604 protein-encoding genes, of which 22.2% have no clear homologues in known genomes. To date, this draft genome is the first one ever determined for an axenic cyanobacterium from Antarctica.
ABSTRACT
The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background.
Subject(s)
Anti-Bacterial Agents/pharmacology , Phenotype , Selection, Genetic , Staphylococcus aureus/genetics , Vancomycin Resistance/genetics , Vancomycin/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Wall/chemistry , Cell Wall/drug effects , Cell Wall/metabolism , DNA Transposable Elements/drug effects , Gene Expression , Humans , Microbial Sensitivity Tests , Mutation , Penicillin-Binding Proteins/genetics , Penicillin-Binding Proteins/metabolism , Peptidoglycan/metabolism , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Staphylococcus aureus/isolation & purificationABSTRACT
Assuming that bicyclic ß-lactams endowed with high conformational adaptability should more easily form acyl-enzyme complexes with PBP2a than the traditional antibiotics, we have prepared a series of bis-2-oxo-azetidinyl macrocycles as potential inhibitors. The compounds are formally "head-head" (HH) cyclodimers of 1-(ω-alkenoyl)-3-(S)-(ω'-alkenoylamino)-2-azetidinones, with various lengths of the alkene chains, obtained by two successive metathesis reactions using the Grubbs catalyst. All compounds behave as acylating inhibitors of PBP2a and one ß-lactam (5c), embedded into the largest ring (32 atoms), features an activity close to that of Ceftobiprole. Conformational analyses, theoretical reactivity models and docking experiments in PBP2a cavity allow to propose a novel pharmacophore, i.e. the 3-(S)-acylamino-1-acyl-2-azetidinone ring, with the syn-conformation of the imide function, associated to a flexible macrocycle favoring the opening of the active site.
Subject(s)
Enzyme Inhibitors/pharmacology , Macrocyclic Compounds/pharmacology , Methicillin-Resistant Staphylococcus aureus/enzymology , Penicillin-Binding Proteins/antagonists & inhibitors , Peptide Synthases/antagonists & inhibitors , beta-Lactams/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Macrocyclic Compounds/chemical synthesis , Macrocyclic Compounds/chemistry , Models, Molecular , Molecular Conformation , Penicillin-Binding Proteins/metabolism , Peptide Synthases/metabolism , beta-Lactams/chemical synthesis , beta-Lactams/chemistryABSTRACT
Penicillin-binding proteins are a well established, validated and still a very promising target for the design and development of new antibacterial agents. Based on our previous discovery of several noncovalent small-molecule inhibitor hits for resistant PBPs we decided to additionally explore the chemical space around these compounds. In order to clarify their structure-activity relationships for PBP inhibition two new series of compounds were synthesized, characterized and evaluated biochemically: the derivatives of anthranilic acid and naphthalene-sulfonamide derivatives. The target compounds were tested for their inhibitory activities on three different transpeptidases: PBP2a from methicillin-resistant Staphylococcus aureus (MRSA) strains, PBP5fm from Enterococcus faecium strains, and PBP1b from Streptococcus pneumoniae strains. The most promising results for both of these series of compounds were obtained against the PBP2a enzyme with the IC50 values in the micromolar range. Although these results do not represent a significant breakthrough in the field of noncovalent PBP inhibitors, they do provide useful structure-activity relationship data, and thus a more solid basis for the design of potent and noncovalent inhibitors of resistant PBPs.
ABSTRACT
ß-Lactam antibiotics have long been a treatment of choice for bacterial infections since they bind irreversibly to Penicillin-Binding Proteins (PBPs), enzymes that are vital for cell wall biosynthesis. Many pathogens express drug-insensitive PBPs rendering ß-lactams ineffective, revealing a need for new types of PBP inhibitors active against resistant strains. We have identified alkyl boronic acids that are active against pathogens including methicillin-resistant S. aureus (MRSA). The crystal structures of PBP1b complexed to 11 different alkyl boronates demonstrate that in vivo efficacy correlates with the mode of inhibitor side chain binding. Staphylococcal membrane analyses reveal that the most potent alkyl boronate targets PBP1, an autolysis system regulator, and PBP2a, a low ß-lactam affinity enzyme. This work demonstrates the potential of boronate-based PBP inhibitors for circumventing ß-lactam resistance and opens avenues for the development of novel antibiotics that target Gram-positive pathogens.
Subject(s)
Boronic Acids/pharmacology , Cell Wall/drug effects , Drug Design , Penicillin-Binding Proteins/antagonists & inhibitors , Penicillin-Binding Proteins/chemistry , Staphylococcus aureus/drug effects , beta-Lactam Resistance/drug effects , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Boronic Acids/chemistry , Boronic Acids/metabolism , Cell Wall/metabolism , Dose-Response Relationship, Drug , Models, Molecular , Molecular Structure , Penicillin-Binding Proteins/metabolism , Staphylococcus aureus/cytology , Staphylococcus aureus/enzymology , Stereoisomerism , Structure-Activity Relationship , beta-Lactams/chemistry , beta-Lactams/pharmacologyABSTRACT
BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs ß-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for ß-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.