Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which remains a significant global health challenge. The emergence of multidrug-resistant (MDR) Mtb strains imposes the development of new therapeutic strategies. This study focuses on the identification and evaluation of potential inhibitors against Mtb H37Ra through a comprehensive screening of an in-house chemolibrary. Subsequently, a promising pyrimidine derivative (LQM495) was identified as promising and then further investigated by experimental and in silico approaches. In this context, computational techniques were used to elucidate the potential molecular target underlying the inhibitory action of LQM495. Then, a consensus reverse docking (CRD) protocol was used to investigate the interactions between this compound and several Mtb targets. Out of 98 Mtb targets investigated, the enhanced intracellular survival (Eis) protein emerged as a target for LQM495. To gain insights into the stability of the LQM495-Eis complex, molecular dynamics (MD) simulations were conducted over a 400 ns trajectory. Further insights into its binding modes within the Eis binding site were obtained through a Quantum mechanics (QM) approach, using density functional theory (DFT), with B3LYP/D3 basis set. These calculations shed light on the electronic properties and reactivity of LQM495. Subsequently, inhibition assays and kinetic studies of the Eis activity were used to investigate the activity of LQM495. Then, an IC50 value of 11.0 ± 1.4 µM was found for LQM495 upon Eis protein. Additionally, its Vmax, Km, and Ki parameters indicated that it is a competitive inhibitor. Lastly, this study presents LQM495 as a promising inhibitor of Mtb Eis protein, which could be further explored for developing novel anti-TB drugs in the future.

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Thermostable l-glutamate oxidases (LGOXs) are desirable for use in l-glutamate (L-Glu) assay kits, enzymatic synthesis of α-ketoglutarate and for biosensor development. However, protein engineering efforts to improve thermostability often lead to a decrease in enzymatic activity. In this report, we aimed to enhance the thermostability (melting temperature, Tm) of a mesophilic LGOX from Streptomyces sp. NT1 (LGOXNT1) without a reduction in activity by a sequence-based protein design approach, termed full consensus (Fc) protein design. Among the 690 amino acids of LGOXNT1, 104 amino acids were substituted by the Fc protein design. The mutant gene was artificially synthesized and expressed in Escherichia coli BL21(DE3) cells. The Tm of the purified, recombinant LGOX mutant (FcLGOX) was determined to be ∼72 °C, which is an increase on the Tm of 65 °C for LGOXNT1 and the highest among known LGOXs. Importantly, purified FcLGOX showed no loss of specific activity or substrate specificity after a 30-min incubation at 70 °C. Our findings provide a new approach to improve the thermostability of enzymes.

Consensus design (CD) is a representative sequence-based protein design method that enables the design of highly functional proteins by analyzing vast amounts of protein sequence data. This study proposes a partial consensus design (PCD) of a protein as a derivative approach of CD. The method replaces the target protein sequence with a consensus sequence in a secondary-structure-dependent manner (i.e., regionally dependent and divided into α-helix, β-sheet, and loop regions). In this study, we generated several artificial partial consensus l-threonine 3-dehydrogenases (PcTDHs) by PCD using the TDH from Cupriavidus necator (CnTDH) as a target protein. Structural and functional analysis of PcTDHs suggested that thermostability would be independently improved when consensus mutations are introduced into the loop region of TDHs. On the other hand, enzyme kinetic parameters (kcat/Km) and average productivity would be synergistically enhanced by changing the combination of the mutations-replacement of one region of CnTDH with a consensus sequence provided only negative effects, but the negative effects were nullified when the two regions were replaced simultaneously. Taken together, we propose the hypothesis that there are protein regions that encode individual protein properties, such as thermostability and activity, and that the introduction of consensus mutations into these regions could additively or synergistically modify their functions.

Cytochrome P450 enzymes have attracted much interest over the years given their ability to insert oxygen into saturated carbon-hydrogen bonds, a difficult feat to accomplish by traditional chemistry. Much of the activity in this field has centered on the bacterial enzyme CYP102A1, or BM3, from Bacillus megaterium, as it has shown itself capable of hydroxylating/acting upon a wide range of substrates, thereby producing industrially relevant pharmaceuticals, fine chemicals, and hormones. In addition, unlike most cytochromes, BM3 is both soluble and fused to its natural redox partner, thus facilitating its use. The industrial use of BM3 is however stifled by its instability and its requirement for the expensive NADPH cofactor. In this work, we added several mutations to the BM3 mutant R966D/W1046S that enhanced the turnover number achievable with the inexpensive cofactors NADH and NBAH. These new mutations, A769S, S847G, S850R, E852P, and V978L, are localized on the reductase domain of BM3 thus leaving the oxidase domain intact. For NBAH-driven reactions by new mutant NTD5, this led to a 5.24-fold increase in total product output when compared to the BM3 mutant R966D/W1046S. For reactions driven by NADH by new mutant NTD6, this enhanced total product output by as much as 2.3-fold when compared to the BM3 mutant R966D/W1046S. We also demonstrated that reactions driven by NADH with the NTD6 mutant not only surpassed total product output achievable by wild-type BM3 with NADPH but also retained the ability to use this latter cofactor with greater total product output as well.

Exponentially increasing protein sequence data enables artificial enzyme design using sequence-based protein design methods, including full-consensus protein design (FCD). The success of artificial enzyme design is strongly dependent on the nature of the sequences used. Hence, sequences must be selected from databases and curated libraries prepared to enable a successful design by FCD. In this study, we proposed a selection approach regarding several key residues as sequence motifs. We used l-threonine 3-dehydrogenase (TDH) as a model to test the validity of this approach. In the classification, four residues (143, 174, 188, and 214) were used as key residues. We classified thousands of TDH homologous sequences into five groups containing hundreds of sequences. Utilizing sequences in the libraries, we designed five artificial TDHs by FCD. Among the five, we successfully expressed four in soluble form. Biochemical analysis of artificial TDHs indicated that their enzymatic properties vary; half of the maximum measured enzyme activity (t1/2) and activation energies were distributed from 53 to 65 °C and from 38 to 125 kJ/mol, respectively. The artificial TDHs had unique kinetic parameters, distinct from one another. Structural analysis indicates that consensus mutations are mainly introduced in the secondary or outer shell. The functional diversity of the artificial TDHs is due to the accumulation of mutations that affect their physicochemical properties. Taken together, our findings indicate that our proposed approach can help generate artificial enzymes with unique enzymatic properties.

Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme\'s stability while at the same time enhancing the enzyme\'s activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments.

Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniae cop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd ) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs.IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.

Coagulase-negative staphylococci (CoNS) are frequently isolated in clinical specimens and are important reservoirs of resistance genes. In 2019, the Brazilian government set the BrCAST/EUCAST (Brazilian Committee on Antimicrobial Susceptibility Testing) guidelines as the national standard, resulting in changes in the interpretation of CoNS susceptibility tests. From outpatients, disk-diffusion susceptibility of 65 CoNS cultures were evaluated and compared using classification criteria from both CLSI and BrCAST/EUCAST. The isolates were identified using matrix assisted laser desorption ionization-time of flight (MALDI-TOF), and the presence of the mecA gene was determined. The most prevalent species were Staphylococcus saprophyticus (32.3%), S. haemolyticus (18.5%), and S. epidermidis (9.2%). Almost perfect agreement was seen between the guidelines, except concerning oxacillin and gentamicin, and the prevalence of multidrug resistant isolates increased with the use of BrCAST/EUCAST. Of all, 15 (23.1%) isolates, mainly S. epidermidis and S. haemolyticus, were positive for the mecA gene, and only three were detected when using CLSI or BrCAST/EUCAST disk-diffusion screening. This, using either guideline, could reveal the difficulty of determining oxacillin resistance. Using warning zones or molecular methods might well be indicated for CoNS. In conclusion, adoption of the BrCAST/EUCAST guidelines will result in certain artificial changes in epidemiological susceptibility profiles, and clinicians and institutions should be aware of the possible implications.

Consensus-based protein engineering strategy has been applied to various proteins and it can lead to the design of proteins with enhanced biological performance. Histone-like HUs comprise a protein family with sequence variety within a highly conserved 3D-fold. HU function includes compacting and regulating bacterial DNA in a wide range of biological conditions in bacteria. To explore the possible impact of consensus-based design in the thermodynamic stability of HU proteins, the approach was applied using a dataset of sequences derived from a group of 40 mesostable, thermostable, and hyperthermostable HUs. The consensus-derived HU protein was named HUBest, since it is expected to perform best. The synthetic HU gene was overexpressed in E. coli and the recombinant protein was purified. Subsequently, HUBest was characterized concerning its correct folding and thermodynamic stability, as well as its ability to interact with plasmid DNA. A substantial increase in HUBest stability at high temperatures is observed. HUBest has significantly improved biological performance at ambience temperature, presenting very low Kd values for binding plasmid DNA as indicated from the Gibbs energy profile of HUBest. This Kd may be associated to conformational changes leading to decreased thermodynamic stability and, therefore, higher flexibility at ambient temperature.