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Journal of Molecular Biology Jul 2020Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and...
Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity.
Topics: Bacterial Proteins; Catalysis; Enzyme Activation; Enzyme Stability; Escherichia coli; Firmicutes; Models, Molecular; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases (Nitrogenous Group Acceptor); Protein Conformation; Protein Domains; Protein Engineering; Recombinant Fusion Proteins; Thermodynamics
PubMed: 32504625
DOI: 10.1016/j.jmb.2020.05.024 -
The ISME Journal Jun 2023Carbohydrate utilization is critical to microbial survival. The phosphotransferase system (PTS) is a well-documented microbial system with a prominent role in...
Carbohydrate utilization is critical to microbial survival. The phosphotransferase system (PTS) is a well-documented microbial system with a prominent role in carbohydrate metabolism, which can transport carbohydrates through forming a phosphorylation cascade and regulate metabolism by protein phosphorylation or interactions in model strains. However, those PTS-mediated regulated mechanisms have been underexplored in non-model prokaryotes. Here, we performed massive genome mining for PTS components in nearly 15,000 prokaryotic genomes from 4,293 species and revealed a high prevalence of incomplete PTSs in prokaryotes with no association to microbial phylogeny. Among these incomplete PTS carriers, a group of lignocellulose degrading clostridia was identified to have lost PTS sugar transporters and carry a substitution of the conserved histidine residue in the core PTS component, HPr (histidine-phosphorylatable phosphocarrier). Ruminiclostridium cellulolyticum was then selected as a representative to interrogate the function of incomplete PTS components in carbohydrate metabolism. Inactivation of the HPr homolog reduced rather than increased carbohydrate utilization as previously indicated. In addition to regulating distinct transcriptional profiles, PTS associated CcpA (Catabolite Control Protein A) homologs diverged from previously described CcpA with varied metabolic relevance and distinct DNA binding motifs. Furthermore, the DNA binding of CcpA homologs is independent of HPr homolog, which is determined by structural changes at the interface of CcpA homologs, rather than in HPr homolog. These data concordantly support functional and structural diversification of PTS components in metabolic regulation and bring novel understanding of regulatory mechanisms of incomplete PTSs in cellulose-degrading clostridia.
Topics: Bacterial Proteins; Cellulose; Histidine; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases; Carbohydrates; Firmicutes; DNA
PubMed: 36899058
DOI: 10.1038/s41396-023-01392-2 -
Protein Science : a Publication of the... Aug 2010Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic...
Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2''-IVa (APH(2'')-IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi-Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2'')-IVa enzyme phosphorylates various 4,6-disubstituted aminoglycoside antibiotics with catalytic efficiencies (k(cat)/K(m)) of 1.5 x 10(3) to 1.2 x 10(6) (M(-1) s(-1)). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2'')-IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP-utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP-based enzyme inhibitors which would not be expected to interfere with crucial ATP-dependent enzymes.
Topics: Adenosine Triphosphate; Aminoglycosides; Binding Sites; Carbohydrate Conformation; Carbohydrate Sequence; Crystallography, X-Ray; Guanosine Triphosphate; Models, Molecular; Molecular Sequence Data; Molecular Structure; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 20556826
DOI: 10.1002/pro.437 -
Journal of Bacteriology May 2023The bacterial nitrogen-related phosphotransfer (PTS; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and...
The bacterial nitrogen-related phosphotransfer (PTS; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and phosphorylation. The Nitro-PTS comprises an enzyme I (EI), PtsP; an intermediate phosphate carrier, PtsO; and a terminal acceptor, PtsN, which is thought to exert regulatory effects that depend on its phosphostate. For instance, biofilm formation by Pseudomonas aeruginosa can be impacted by the Nitro-PTS, as deletion of either or suppresses Pel exopolysaccharide production and additional deletion of elevates Pel production. However, the phosphorylation state of PtsN in the presence and absence of its upstream phosphotransferases has not been directly assessed, and other targets of PtsN have not been well defined in P. aeruginosa. We show that PtsN phosphorylation via PtsP requires the GAF domain of PtsP and that PtsN is phosphorylated on histidine 68, as in Pseudomonas putida. We also find that FruB, the fructose EI, can substitute for PtsP in PtsN phosphorylation but only in the absence of PtsO, implicating PtsO as a specificity factor. Unphosphorylatable PtsN had a minimal effect on biofilm formation, suggesting that it is necessary but not sufficient for the reduction of Pel in a deletion. Finally, we use transcriptomics to show that the phosphostate and the presence of PtsN do not appear to alter the transcription of biofilm-related genes but do influence genes involved in type III secretion, potassium transport, and pyoverdine biosynthesis. Thus, the Nitro-PTS influences several P. aeruginosa behaviors, including the production of its signature virulence factors. The PtsN protein impacts the physiology of a number of bacterial species, and its control over downstream targets can be altered by its phosphorylation state. Neither its upstream phosphotransferases nor its downstream targets are well understood in Pseudomonas aeruginosa. Here, we examine PtsN phosphorylation and find that the immediate upstream phosphotransferase acts as a gatekeeper, allowing phosphorylation by only one of two potential upstream proteins. We use transcriptomics to discover that PtsN regulates the expression of gene families that are implicated in virulence. One emerging pattern is a repression hierarchy by different forms of PtsN: its phosphorylated state is more repressive than its unphosphorylated state, but the expression of its targets is even higher in its complete absence.
Topics: Bacterial Proteins; Pseudomonas aeruginosa; Virulence; Phosphorylation; Phosphotransferases; Phosphoenolpyruvate Sugar Phosphotransferase System; Gene Expression Regulation, Bacterial
PubMed: 37074168
DOI: 10.1128/jb.00453-22 -
The Journal of Biological Chemistry Mar 2008Aminoglycoside 2''-phosphotransferases mediate high level resistance to aminoglycoside antibiotics in Gram-positive microorganisms, thus posing a serious threat to the...
Aminoglycoside 2''-phosphotransferases mediate high level resistance to aminoglycoside antibiotics in Gram-positive microorganisms, thus posing a serious threat to the treatment of serious enterococcal infections. This work reports on cloning, purification, and detailed mechanistic characterization of aminoglycoside 2''-phosphotransferase, known as type Ic enzyme. In an unexpected finding, the enzyme exhibits strong preference for guanosine triphosphate over adenosine triphosphate as the phosphate donor, a unique observation among all characterized aminoglycoside phosphotransferases. The enzyme phosphorylates only certain 4,6-disubstituted aminoglycosides exclusively at the 2''-hydroxyl with k(cat) values of 0.5-1.0 s(-1) and K(m) values in the nanomolar range for all substrates but kanamycin A. Based on this unique substrate profile, the enzyme is renamed aminoglycoside 2''-phosphotransferase type IIIa. Product and dead-end inhibition patterns indicated a random sequential Bi Bi mechanism. Both the solvent viscosity effect and determination of the rate constant for dissociation of guanosine triphosphate indicated that at pH 7.5 the release of guanosine triphosphate is rate-limiting. A computational model for the enzyme is presented that sheds light on the structural aspects of interest in this family of enzymes.
Topics: Adenosine Triphosphate; Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Catalysis; Cloning, Molecular; Computer Simulation; Drug Resistance, Bacterial; Enterococcus; Gram-Positive Bacterial Infections; Guanosine Triphosphate; Models, Chemical; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Substrate Specificity
PubMed: 18199745
DOI: 10.1074/jbc.M709645200 -
Frontiers in Cellular and Infection... 2021The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the gene, is a potential drug target for pathogenic fungi, such as . However, both...
The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the gene, is a potential drug target for pathogenic fungi, such as . However, both substrate-binding sites of Cho1 have not been characterized. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif, which is present within Cho1. We tested the CAPT motif for its role in PS synthesis by mutating conserved residues using alanine substitution mutagenesis. PS synthase assays revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1 function. In contrast, there were no clear motifs in Cho1 for binding serine. Therefore, to identify the serine binding site, PS synthase sequences from three fungi were aligned with sequences of a similar enzyme, phosphatidylinositol (PI) synthase, from the same fungi. This revealed a motif that was unique to PS synthases. Using alanine substitution mutagenesis, we found that some of the residues in this motif are required for Cho1 function. Two alanine substitution mutants, L184A and R189A, exhibited contrasting impacts on PS synthase activity, and were characterized for their Michaelis-Menten kinetics. The L184A mutant displayed enhanced PS synthase activity and showed an increased . In contrast, R189A showed decreased PS synthase activity and increased for serine, suggesting that residue R189 is involved in serine binding. These results help to characterize PS synthase substrate binding, and should direct rational approaches for finding Cho1 inhibitors that may lead to better antifungals.
Topics: Binding Sites; CDPdiacylglycerol-Serine O-Phosphatidyltransferase; Candida albicans; Phosphotransferases; Saccharomyces cerevisiae
PubMed: 35004345
DOI: 10.3389/fcimb.2021.765266 -
The Biochemical Journal Jul 1974The activity of a number of alcohols was examined as substrates or inhibitors of glycerol kinase (ATP-glycerol phosphotransferase; EC 2.7.1.30) from Candida mycoderma....
The activity of a number of alcohols was examined as substrates or inhibitors of glycerol kinase (ATP-glycerol phosphotransferase; EC 2.7.1.30) from Candida mycoderma. On the basis of these and other results, a modified model is proposed to account for the substrate specificity of the enzyme.
Topics: Adenosine Triphosphate; Alcohols; Candida; Glycerol; L-Lactate Dehydrogenase; Models, Biological; NAD; Phosphoenolpyruvate; Phosphotransferases; Pyruvate Kinase; Structure-Activity Relationship; Sugar Alcohols; Tetroses
PubMed: 4375973
DOI: 10.1042/bj1410305 -
Journal of Microbiology and... Aug 2022When , a glucose-specific phosphotransferase system (PTS) component, is deleted in , growth can be severely poor because of the lack of efficient glucose transport. We...
When , a glucose-specific phosphotransferase system (PTS) component, is deleted in , growth can be severely poor because of the lack of efficient glucose transport. We discovered a new PTS transport system that could transport glucose through the growth-coupled experimental evolution of -deficient C strain under anaerobic conditions. Genome sequencing revealed mutations in , which encodes a repressor of -acetylgalactosamine (Aga) PTS expression in evolved progeny strains. RT-qPCR analysis showed that the expression of Aga PTS gene increased because of the loss-of-function of . We confirmed the efficient Aga PTS-mediated glucose uptake by genetic complementation and anaerobic fermentation. We discussed the discovery of new glucose transporter in terms of different genetic backgrounds of strains, and the relationship between the pattern of mixed-acids fermentation and glucose transport rate.
Topics: Acetylgalactosamine; Agar; Escherichia coli; Escherichia coli Proteins; Glucose; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases
PubMed: 35791075
DOI: 10.4014/jmb.2205.05059 -
Proteins Apr 2013The crystal structure of Ta0880, determined at 1.91 Å resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β/α sandwich...
The crystal structure of Ta0880, determined at 1.91 Å resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β/α sandwich domain and a smaller lid domain. The overall fold belongs to the PfkB family of carbohydrate kinases (a family member of the Ribokinase clan) which include ribokinases, 1-phosphofructokinases, 6-phosphofructo-2-kinase, inosine/guanosine kinases, fructokinases, adenosine kinases, and many more. Based on its general fold, Ta0880 had been annotated as a ribokinase-like protein. Using a coupled pyruvate kinase/lactate dehydrogenase assay, the activity of Ta0880 was assessed against a variety of ribokinase/pfkB-like family substrates; activity was not observed for ribose, fructose-1-phosphate, or fructose-6-phosphate. Based on structural similarity with nucleoside kinases (NK) from Methanocaldococcus jannaschii (MjNK, PDB 2C49, and 2C4E) and Burkholderia thailandensis (BtNK, PDB 3B1O), nucleoside kinase activity was investigated. Ta0880 (TaNK) was confirmed to have nucleoside kinase activity with an apparent KM for guanosine of 0.21 μM and catalytic efficiency of 345,000 M(-1) s(-1) . These three NKs have significantly different substrate, phosphate donor, and cation specificities and comparisons of specificity and structure identified residues likely responsible for the nucleoside substrate selectivity. Phylogenetic analysis identified three clusters within the PfkB family and indicates that TaNK is a member of a new sub-family with broad nucleoside specificities. Proteins 2013. © 2012 Wiley Periodicals, Inc.
Topics: Amino Acid Sequence; Burkholderia; Crystallography, X-Ray; Kinetics; Methanococcales; Molecular Sequence Data; Phosphotransferases; Protein Multimerization; Protein Structure, Secondary; Sequence Alignment; Substrate Specificity; Thermoplasma
PubMed: 23161756
DOI: 10.1002/prot.24212 -
Acta Crystallographica. Section F,... Jun 2012Adenylate kinases (AKs) are phosphotransferase enzymes that catalyze the interconversion of adenine nucleotides, thereby playing an important role in energy metabolism....
Adenylate kinases (AKs) are phosphotransferase enzymes that catalyze the interconversion of adenine nucleotides, thereby playing an important role in energy metabolism. In Plasmodium falciparum, three AK isoforms, namely PfAK1, PfAK2 and GTP:AMP phosphotransferase (PfGAK), have been identified. While PfAK1 and PfAK2 catalyse the conversion of ATP and AMP to two molecules of ADP, PfGAK exhibits a substrate preference for GTP and AMP and does not accept ATP as a substrate. PfGAK was cloned and expressed in Escherichia coli and purified using two-step chromatography. Brown hexagonal crystals of PfGAK were obtained and a preliminary diffraction analysis was performed. X-ray diffraction data for a single PfGAK crystal were processed to 2.9 Å resolution in space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 123.49, c = 180.82 Å, α = β = 90, γ = 120°.
Topics: Crystallization; Crystallography, X-Ray; Gene Expression; Phosphotransferases (Phosphate Group Acceptor); Plasmodium falciparum
PubMed: 22684067
DOI: 10.1107/S1744309112015862