Biotin Protein Ligase Research Articles

An ATP detection system based on the enzyme reaction with biotin protein ligase

Adenosine triphosphate (ATP) is the energy currency of all living organisms and can be used as an indicator for cell proliferation and cytotoxicity. In the present work, we have developed a novel ATP detection system by combining the biotinylation reaction from archaeon Sulfolobus tokodaii with fluorescence resonance energy transfer (FRET). In biotinylation from S. tokodaii, an enzyme known as biotin protein ligase (BPL) forms a very stable complex with its product, biotinylated substrate protein (BCCP). Here, BPL and BCCP were fused to the fluorescent proteins Cerulean and Clover, respectively, and ATP detection was accomplished by monitoring the FRET signal between the two fluorescent proteins, since ATP is an essential component for biotinylation and the tight BPL-BCCP complex is formed only after biotinylation. Using this system, we have succeeded in detecting 5 nM of ATP by biotinylation reaction with 50 nM of each fusion protein. Our method has a characteristic that the signal does not decay for at least 2 h after the start of the reaction, unlike in the case of the luminescence-based assay with luciferase commonly used for the ATP detection. Thus, our system allows for ATP detection which is not significantly constrained by measurement timing.

New Potent Sulfonamide-Based Inhibitors of S. aureus Biotin Protein Ligase.

The key regulatory metabolic enzyme, biotin protein ligase (BPL), is an attractive target for the development of novel antibiotics against multi-drug-resistant bacteria, such as Staphylococcus aureus. Here we report the synthesis and assay of a new series of inhibitors (6-9) against S. aureus BPL (SaBPL), where a component sulfonamide linker was used to mimic the acyl-phosphate group of the natural intermediate biotinyl-5'-AMP (1). A pivotal correlation between the acidity of the central NH of the sulfonamide linker of 6-9 and in vitro inhibitory activity against SaBPL was observed. Specifically, sulfonylcarbamate 8, with its highly acidic sulfonyl central NH, as evaluated by 1H NMR spectroscopy, showed exceptional potency (K i = 10.3 ± 3.8 nM). Furthermore, three inhibitors demonstrated minimum inhibitory concentrations of 16-32 μg/mL against clinical methicillin-resistant S. aureus (MRSA) strains.

Structural insights into BirA from Haemophilus influenzae, a bifunctional protein as a biotin protein ligase and a transcriptional repressor

Biotin is an essential coenzyme involved in various metabolic processes across all known organisms, with biotinylation being crucial for the activity of carboxylases. BirA from Haemophilus influenzae is a bifunctional protein that acts as a biotin protein ligase and a transcriptional repressor. This study reveals the crystal structures of Hin BirA in both its apo- and holo-(biotinyl-5′-AMP bound) forms. As a class II BirA, it consists of three domains: N-terminal DNA binding domain, central catalytic domain, and C-terminal SH3-like domain. The structural analysis shows that the biotin-binding loop forms an ordered structure upon biotinyl-5′-AMP binding. This facilitates its interaction with the ligand and promotes protein dimerization. Comparative studies with other BirA homologs from different organisms indicate that the residues responsible for binding biotinyl-5′-AMP are highly conserved. This study also utilized AlphaFold2 to model the potential heterodimeric interaction between Hin BirA and biotin carboxyl carrier protein, thereby providing insights into the structural basis for biotinylation. These findings enhance our understanding of the structural and functional characteristics of Hin BirA, highlighting its potential as a target for novel antibiotics that disrupt the bacterial biotin synthesis pathways.

Chemical Constituents, Antioxidant, and Antimicrobial Activities of Ethyl Acetate Fractionated Extract from Rhizomes of Zingiber monophyllum Gagnep.: In vitro and in silico Screenings

Objective/Background: Zingiber monophyllum Gagnep., a member of the Zingiberaceae family, is known for its significant biological activities. The current study aimed to determine the volatile components of the ethyl acetate (EtOAc) fractionated extract found in the rhizomes of this species. This is the first report on the chemical composition and bioactivities of Z. monophyllum rhizomes fractionated extract. Methods: The chemical constituents were analyzed and determined using gas chromatography-mass spectrometry (GC-MS). Antioxidant activities were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power (FRAP) assays using ascorbic acid as a positive control. Antibacterial and antifungal properties of the EtOAc fractionated extract of Z. monophyllum rhizomes were assessed against Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus, and Candida albicans. Density functional theory (DFT) and molecular docking were also employed to illustrate antioxidant and antimicrobial activities. Results: Nine components were identified by GC-MS analysis from the EtOAc fractionated extract of Z. monophyllum rhizomes. ( E)-labda-8(17),12-diene-15,16-dial (9), spathulenol (2), and neointermedeol (5) were the major components (21.8%, 16.8%, and 11.9%, respectively). Moderate antioxidant activities of the EtOAc fractionated extract were observed via both the DPPH assay and the FRAP assay using ascorbic acid as the standard compound. The extract demonstrated remarkable antimicrobial activity against all examined microbial strains, except for P. aeruginosa. The DFT study analyzed the antioxidant potential of each component in the fractionated extract. Molecular docking study chose E. faecalis DNA gyrase B, E. coli DNA gyrase B, S. aureus biotin protein ligase, E. faecalis Alanine racemase, and C. albicans N-myristoyltransferase as potential target proteins for antimicrobial activity. Conclusion: In this study, the chemical composition of the EtOAc fractionated extract of Z monophyllum rhizomes was demonstrated through GC-MS analysis for the first time. Nine components, including alloaromadendrene, spathulenol, globulol, τ-cadinol, neointermedeol, aromadendrene oxide-(2), ambrial, (E)-15,16-dinorlabda-8(17),11-dien-13-one, and (E)-lambda-8 (17),12-diene-15,16-dial along with relative content were identified in this fractionated extract. The bioassays revealed that the fractionated extract showed moderate antioxidant activities and significant antimicrobial activities. The antioxidant and antimicrobial potential of each component was also theoretically examined by the DFT study and molecular docking study, respectively.

The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

Repurposing Proximity-Dependent Protein Labeling (BioID2) for Protein Interaction Mapping in E. coli.

Methods that identify protein-protein interactions are essential for understanding molecular mechanisms controlling biological systems. Proximity-dependent labeling has proven to be a valuable method for revealing protein-protein interaction networks in living cells. A mutant form of the biotin protein ligase enzyme from Aquifex aeolicus (BioID2) underpins this methodology by producing biotin that is attached to proteins that enter proximity to it. This labels proteins for capture, extraction, and identification. In this chapter, we present a toolkit for BioID2 specifically adapted for use in E. coli, exemplified by the chemotaxis protein CheA. We have created plasmids containing BioID2 as expression cassettes for proteins (e.g., CheA) fused to BioID2 at either the N or C terminus, optimized with an 8×GGS linker. We provide a methodology for expression and verification of CheA-BioID2 fusion proteins in E. coli cells, the in vivo biotinylation of interactors by protein-BioID2 fusions, and extraction and analysis of interacting proteins that have been biotinylated.

Synthesis, Antimicrobial and Computational Studies of New Branched Azaphenothiazinones Derivatives

In a continued search for new medicinally active nonlinear phenothiazines, novel angular chloroazaphenothiazinone derivatives have been synthesized via transition metal-catalyzed cross-coupling reactions. The structural elucidation of the synthesized compounds was established by a combined spectroscopic and elemental analysis. The synthesized compounds were tested for their antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Enterococusfaecalis, Escherichia coli, Candida albican,and Aspergillus niger isolates by the convectional agar-well dilution method and compounds 5c and 8cdisclosed excellent in vitro activity against some of the tested microorganisms. In silico,the study showed that the synthesized compounds possessed promising physichemical properties and fit well in the active site of a Biotin-Protein Ligase (BPL) forming essential hydrogen bonding and hydrophobic interactions.

Biotin protein ligase as you like it: Either extraordinarily specific or promiscuous protein biotinylation.

Biotin (vitamin H or B7) is a coenzyme essential for all forms of life. Biotin has biological activity only when covalently attached to a few key metabolic enzyme proteins. Most organisms have only one attachment enzyme, biotin protein ligase (BPL), which attaches biotin to all target proteins. The sequences of these proteins and their substrate proteins are strongly conserved throughout biology. Structures of both the biotin ligase- and biotin-acceptor domains of mammals, plants, several bacterial species, and archaea have been determined. These, together with mutational analyses of ligases and their protein substrates, illustrate the exceptional specificity of this protein modification. For example, the Escherichia coli BPL biotinylates only one of the >4000 cellular proteins. Several bifunctional bacterial biotin ligases transcriptionally regulate biotin synthesis and/or transport in concert with biotinylation. The human BPL has been demonstrated to play an important role in that mutations in the BPL encoding gene cause one form of the disease, biotin-responsive multiple carboxylase deficiency. Promiscuous mutant versions of several BPL enzymes release biotinoyl-AMP, the active intermediate of the ligase reaction, to solvent. The released biotinoyl-AMP acts as a chemical biotinylation reagent that modifies lysine residues of neighboring proteins in vivo. This proximity-dependent biotinylation (called BioID) approach has been heavily utilized in cell biology.

Emergence of allostery through reorganization of protein residue network architecture.

Despite more than a century of study, consensus on the molecular basis of allostery remains elusive. A comparison of allosteric and non-allosteric members of a protein family can shed light on this important regulatory mechanism, and the bacterial biotin protein ligases, which catalyze post-translational biotin addition, provide an ideal system for such comparison. While the Class I bacterial ligases only function as enzymes, the bifunctional Class II ligases use the same structural architecture for an additional transcription repression function. This additional function depends on allosterically activated homodimerization followed by DNA binding. In this work, we used experimental, computational network, and bioinformatics analyses to uncover distinguishing features that enable allostery in the Class II biotin protein ligases. Experimental studies of the Class II Escherichia coli protein indicate that catalytic site residues are critical for both catalysis and allostery. However, allostery also depends on amino acids that are more broadly distributed throughout the protein structure. Energy-based community network analysis of representative Class I and Class II proteins reveals distinct residue community architectures, interactions among the communities, and responses of the network to allosteric effector binding. Bioinformatics mutual information analyses of multiple sequence alignments indicate distinct networks of coevolving residues in the two protein families. The results support the role of divergent local residue community network structures both inside and outside of the conserved enzyme active site combined with distinct inter-community interactions as keys to the emergence of allostery in the Class II biotin protein ligases.

Structural Study of Potent Triazole-Based Inhibitors of Staphylococcus aureus Biotin Protein Ligase.

The rise of multidrug-resistant bacteria, such as Staphylococcus aureus, has highlighted global urgency for new classes of antibiotics. Biotin protein ligase (BPL), a critical metabolic regulatory enzyme, is an important target that shows significant promise in this context. Here we report the in silico docking, synthesis, and biological assay of a new series of N1-diphenylmethyl-1,2,3-triazole-based S. aureus BPL (SaBPL) inhibitors (8-19) designed to probe the adenine binding site and define whole-cell activity for this important class of inhibitor. Triazoles 13 and 14 with N1-propylamine and -butanamide substituents, respectively, were particularly potent with K i values of 10 ± 2 and 30 ± 6 nM, respectively, against SaBPL. A strong correlation was apparent between the K i values for 8-19 and the in silico docking, with hydrogen bonding to amino acid residues S128 and N212 of SaBPL likely contributing to potent inhibition.

Understanding BirA allostery from a network perspective using MD simulations.

Allostery is how two faraway sites communicate to affect their functionality in a biomacromolecule. It is integral to cellular processes like enzymatic catalysis, signal transduction, metabolism, and transcription regulation. The physical manifestations of subtle perturbations, such as amino acid substitutions, on a faraway location can guide us to rationally design allosteric drugs. To explore the effects of remote mutations on a functional site, we work on an allosteric system namely, Biotin Protein Ligase (BPL) extracted from eukaryotic Escherichia coli. This bifunctional BPL allosterically controls homodimerization to bind DNA and repress transcription initiation, as well as catalyze biotin linkage to biotin-dependent carboxylases. Previous thermodynamic studies on various E.coli BPL variants explored the tunability of allosterically modulated homodimerization by performing amino acid substitutions distant from the dimerization site (ranging from weak dimerizers to superdimerizers). In this work, we characterized distinct responses of energy-based protein network architectures to these mutations using Molecular Dynamics simulations and implementing tools from Network theory. Results indicate that the inflow of energetic information to the dimerization site in a protein is correlated to its homodimerization property. Thus, mutations affect functional activity in operating sites by redirecting information traffic in protein residue networks.

Synthesis of novel tetrazolic derivatives and evaluation of their antimicrobial activity

The microbial resistance to various anti-infective drugs remains a major worldwide health problem. Therefore, the search for novel and effective antimicrobial agents is of paramount importance to combat these pathogenic microorganisms. The present study includes the synthesis of novel Bis(arylidene-2-[(tetrazol-5-yl)]alkane derivatives and the evaluation of their antibacterial and antifungal activities against four bacterial strains (Listeria innocua, Escherichia Coli, Pseudomonas aeruginosa, Staphylococcus aureus) and four fungal strains (Candida albicans, Rhodotorula glutinis, Geotrichum candidum, Aspergillus niger). These tetrazoles derivatives were prepared with moderate yields and their structures were determined on the basis of spectroscopic analyses, including FT IR, NMR, and HR-ESI-MS. Subsequently, these compounds and their precursors were tested in vitro for their antibacterial and antifungal activities, using the agar-disc diffusion method. All synthesized compounds at concentration of 1 mg/ml exhibited some antimicrobial properties against the tested fungal and bacterial strains. Finally, the docking study on the Biotin protein ligase in Staphylococcus aureus, showed that all molecules interact with the active site.

A New 1,2,3-Triazole Scaffold with Improved Potency against Staphylococcus aureus Biotin Protein Ligase.

Staphylococcus aureus, a key ESKAPE bacteria, is responsible for most blood-based infections and, as a result, is a major economic healthcare burden requiring urgent attention. Here, we report in silico docking, synthesis, and assay of N1-diphenylmethyl triazole-based analogues (7-13) designed to interact with the entire binding site of S. aureus biotin protein ligase (SaBPL), an enzyme critical for the regulation of gluconeogenesis and fatty acid biosynthesis. The second aryl ring of these compounds enhances both SaBPL potency and whole cell activity against S. aureus relative to previously reported mono-benzyl triazoles. Analogues 12 and 13, with added substituents to better interact with the adenine binding site, are particularly potent, with Ki values of 6.01 ± 1.01 and 8.43 ± 0.73 nM, respectively. These analogues are the most active triazole-based inhibitors reported to date and, importantly, inhibit the growth of a clinical isolate strain of S. aureus ATCC 49775, with minimum inhibitory concentrations of 1 and 8 μg/mL, respectively.

Screening of marine natural products for potential inhibitors targeting biotin biosynthesis pathway in Mycobacterium tuberculosis

Tuberculosis (TB) remains as one of the major public health concerns worldwide. A successful TB control and treatment is very challenging, due to continuing emergence of Mycobacterium tuberculosis strains resistant to known drugs. Therefore, the development of new drugs with different chemical and biological approaches is necessary to obtain more efficient anti-tubercular therapeutics. Biotin is an essential cofactor for lipid biosynthesis and gluconeogenesis in M. tuberculosis. M. tuberculosis relies on de novo biotin biosynthesis to obtain this vital cofactor since it cannot scavenge sufficient biotin from a mammalian host. In this study, comprehensive in silico methods including structure-based virtual screening, molecular docking, and molecular dynamic simulation analysis for ∼8000 marine natural products were performed against two essential enzymes involved in biotin synthesis and ligation of M. tuberculosis namely, pyridoxal 5′-phosphate-dependent transaminase (BioA) and mycobacterial biotin protein ligase (MtBPL). Two compounds; CMNPD10112 and CMNPD10113 are unveiled to bind the enzymes consistently and with high affinities. The binding pattern of compounds is further noticed in very stable binding modes as analyzed by molecular dynamics simulation and the mean RMSD of the complexes is within 4 Å. The intermolecular binding free energies validated complexes are less than −40 kcal/mol, which demonstrates strong and stable complexes formation. The identified hit compounds could be seeds for design of effective anti-mycobacterium therapeutics by inhibition of bacterial growth through blocking the biotin biosynthesis. Communicated by Ramaswamy H. Sarma

Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.

Two‐way regulation of protein expression for identification and validation of on‐target inhibitors of Mycobacterium tuberculosis

Whole‐cell screens are a powerful tool to identify small molecules that inhibit bacterial growth by targeting pharmacologically relevant mechanisms. Identification of the target for many of these whole cell hits could often be a complicated and time‐consuming process. To supplement Tuberculosis drug discovery effort in this regard, we developed cell‐based assays that utilizes two‐way regulation of protein expression to readily identify on‐target inhibitors of essential genes in Mycobacterium tuberculosis. We successfully used this approach to identify a novel guanidine‐based inhibitor of MmpL3, an essential mycolate transporter in the inner membrane of Mycobacterium tuberculosis from a library of 220 compounds. We next extended this approach to categorize inhibitors that either directly or indirectly impact the activity of Biotin Protein Ligase; BirA. This work highlights the value of target‐based phenotypic screens to identify novel antimycobacterial inhibitors. These assays have significant utility in facilitating prompt identification of new lead molecules and target‐driven optimization of analogues of more advanced scaffolds that inhibit MmpL3 and BirA activity in the cellular context.

Synthesis, antimicrobial activity and in-silico docking of two macrocycles based on pyrazole-tetrazole subunit

The design of two new macrocycles bearing two pyrazole-tetrazole subunits and a lateral arm is presented. These macrocycles and the intermediate compounds are characterized by NMR, FT-IR and UV-Vis spectroscopies as well as elemental analysis and mass spectrometry. Two routes were evaluated to reach this kind of structures in terms of feasibility and reaction yield. The first one was unable to achieve the desired structures and several issues were confronted. However, the second route allows the obtaining of these macrocycles in moderate yields. Their quantum parameters were also determined using DFT calculations. The antimicrobial activity of these macrocycles was evaluated and the obtained results show, that both macrocycles possess good potency to inhibit Staphylococcus aureus. In-silico docking calculations were performed on the biotin protein ligase in Staphylococcus aureus and results suggest a good interactions of macrocycles with the key residues of the active pocket.

Identification of Putative Vaccine and Drug Targets against the Methicillin-Resistant Staphylococcus aureus by Reverse Vaccinology and Subtractive Genomics Approaches.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and responsible for causing life-threatening infections. The emergence of hypervirulent and multidrug-resistant (MDR) S. aureus strains led to challenging issues in antibiotic therapy. Consequently, the morbidity and mortality rates caused by S. aureus infections have a substantial impact on health concerns. The current worldwide prevalence of MRSA infections highlights the need for long-lasting preventive measures and strategies. Unfortunately, effective measures are limited. In this study, we focus on the identification of vaccine candidates and drug target proteins against the 16 strains of MRSA using reverse vaccinology and subtractive genomics approaches. Using the reverse vaccinology approach, 4 putative antigenic proteins were identified; among these, PrsA and EssA proteins were found to be more promising vaccine candidates. We applied a molecular docking approach of selected 8 drug target proteins with the drug-like molecules, revealing that the ZINC4235426 as potential drug molecule with favorable interactions with the target active site residues of 5 drug target proteins viz., biotin protein ligase, HPr kinase/phosphorylase, thymidylate kinase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-L-lysine ligase, and pantothenate synthetase. Thus, the identified proteins can be used for further rational drug or vaccine design to identify novel therapeutic agents for the treatment of multidrug-resistant staphylococcal infection.

Functional dissection and modulation of the BirA protein for improved autotrophic growth of gas-fermenting Clostridiumljungdahlii.

SummaryGas‐fermenting Clostridium species can convert one‐carbon gases (CO2/CO) into a variety of chemicals and fuels, showing excellent application prospects in green biological manufacturing. The discovery of crucial genes and proteins with novel functions is important for understanding and further optimization of these autotrophic bacteria. Here, we report that the Clostridium ljungdahlii BirA protein (ClBirA) plays a pleiotropic regulator role, which, together with its biotin protein ligase (BPL) activity, enables an effective control of autotrophic growth of C. ljungdahlii. The structural modulation of ClBirA, combined with the in vivo and in vitro analyses, further reveals the action mechanism of ClBirA’s dual roles as well as their interaction in C. ljungdahlii. Importantly, an atypical, flexible architecture of the binding site was found to be employed by ClBirA in the regulation of a lot of essential pathway genes, thereby expanding BirA’s target genes to a broader range in clostridia. Based on these findings, molecular modification of ClBirA was performed, and an improved cellular performance of C. ljungdahlii was achieved in gas fermentation. This work reveals a previously unknown potent role of BirA in gas‐fermenting clostridia, providing new perspective for understanding and engineering these autotrophic bacteria.

Genetic models of latent tuberculosis in mice reveal differential influence of adaptive immunity.

Studying latent Mycobacterium tuberculosis (Mtb) infection has been limited by the lack of a suitable mouse model. We discovered that transient depletion of biotin protein ligase (BPL) and thioredoxin reductase (TrxB2) results in latent infections during which Mtb cannot be detected but that relapse in a subset of mice. The immune requirements for Mtb control during latency, and the frequency of relapse, were strikingly different depending on how latency was established. TrxB2 depletion resulted in a latent infection that required adaptive immunity for control and reactivated with high frequency, whereas latent infection after BPL depletion was independent of adaptive immunity and rarely reactivated. We identified immune signatures of T cells indicative of relapse and demonstrated that BCG vaccination failed to protect mice from TB relapse. These reproducible genetic latency models allow investigation of the host immunological determinants that control the latent state and offer opportunities to evaluate therapeutic strategies in settings that mimic aspects of latency and TB relapse in humans.