Conserved Histidine Residues Research Articles

Novel motif associated with carbon catabolite repression in two major Gram-positive pathogen virulence regulatory proteins.

Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phosphotransferase system (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of Streptococcus pyogenes, has an EIIBGat domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of Listeria monocytogenes, harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.IMPORTANCEIn this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification.

Molecular dynamics investigation of the influenza hemagglutinin conformational changes in acidic pH.

The surface protein hemagglutinin (HA) of the influenza virus plays a pivotal role in facilitating viral infection by binding to sialic acid receptors on host cells. Its conformational state is pH-sensitive, impacting its receptor-binding ability and evasion of the host immune response. In this study, we conducted extensive equilibrium microsecond-level all-atom molecular dynamics (MD) simulations of the HA protein to explore the influence of low pH on its conformational dynamics. Specifically, we investigated the impact of protonation on conserved histidine residues (His106 2 ) located in the hinge region of HA2. Our analysis encompassed comparisons between non-protonated (NP), partially protonated (1P, 2P), and fully-protonated (3P) conditions. Our findings reveal substantial pH-dependent conformational alterations in the HA protein, affecting its receptor-binding capability and immune evasion potential. Notably, the non-protonated form exhibits greater stability compared to protonated states. Conformational shifts in the central helices of HA2 involve outward movement, counterclockwise rotation of protonated helices, and fusion peptide release in protonated systems. Disruption of hydrogen bonds between the fusion peptide and central helices of HA2 drives this release. Moreover, HA1 separation is more likely in the fully-protonated system (3P) compared to non-protonated systems (NP), underscoring the influence of protonation. These insights shed light on influenza virus infection mechanisms and may inform the development of novel antiviral drugs targeting HA protein and pH-responsive drug delivery systems for influenza.

A PadR family transcriptional repressor regulates the transcription of chromate efflux transporter in Enterobacter sp. Z1.

Chromium is a prevalent toxic heavy metal, and chromate [Cr(VI)] exhibits high mutagenicity and carcinogenicity. The presence of the Cr(VI) efflux protein ChrA has been identified in strains exhibiting resistance to Cr(VI). Nevertheless, certain strains of bacteria that are resistant to Cr(VI) lack the presence of ChrB, a known regulatory factor. Here, a PadR family transcriptional repressor, ChrN, has been identified as a regulator in the response of Enterobacter sp. Z1(CCTCC NO: M 2019147) to Cr(VI). The chrN gene is cotranscribed with the chrA gene, and the transcriptional expression of this operon is induced by Cr(VI). The binding capacity of the ChrN protein to Cr(VI) was demonstrated by both the tryptophan fluorescence assay and Ni-NTA purification assay. The interaction between ChrN and the chrAN operon promoter was validated by reporter gene assay and electrophoretic mobility shift assay. Mutation of the conserved histidine residues His14 and His50 resulted in loss of ChrN binding with the promoter of the chrAN operon. This observation implies that these residues are crucial for establishing a DNA-binding site. These findings demonstrate that ChrN functions as a transcriptional repressor, modulating the cellular response of strain Z1 to Cr(VI) exposure.

Two Toxoplasma gondii putative pore-forming proteins, GRA47 and GRA72, influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the PVM, which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVM's permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47 or those with a histidine mutation have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against toxoplasmosis.

Mutation of conserved histidine residues of dengue virus envelope protein impairs viral like particle maturation and secretion

Dengue virus (DENV) envelope protein plays crucial role in virus entry and maturation of virus during infection. Maturation of DENV occurs in the trans Golgi network at slightly acidic pH which is close to pKa of histidine. When exposed to the acidic environment of the late secretory pathway, dengue virus particles go through a significant conformational change, whereby interactions of structural proteins envelope (E) and prM proteins are reorganised and enable furin protease to cleave prM resulting in mature virus. In order to study the role of histidine of E protein in DENV maturation, we mutated 7 conserved histidine residues of envelope protein and assessed the percent of budding using viral like particle (VLP) system. Histidine mutants; H144A, H244A, H261A and H282A severely disrupted VLP formation without any significant change in expression in cell and its oligomerization ability. Treatment with acidotropic amine reversed the defect for all 4 mutants suggesting that these histidines could be involved in maturation and release. Over expression of capsid protein slightly enhanced VLP release of H244A and H261A. Similarly, furin over expression increased VLP release of these mutants. Co-immunoprecipitation studies revealed that prM and E interaction is lost for H244A, H261A and H282A mutants at acidic pH but not at neutral pH indicating that they could be involved in histidine switch during maturation at acidic pH. Detailed analysis of the mutants could provide novel insights on the interplay of envelop protein during maturation and aid in target for drug development.

Catalytic Mechanism of Mycobacterium tuberculosis Methionine Sulfoxide Reductase A.

The oxidation of Met to methionine sulfoxide (MetSO) by oxidants such as hydrogen peroxide, hypochlorite, or peroxynitrite has profound effects on protein function. This modification can be reversed by methionine sulfoxide reductases (msr). In the context of pathogen infection, the reduction of oxidized proteins gains significance due to microbial oxidative damage generated by the immune system. For example, Mycobacterium tuberculosis (Mt) utilizes msrs (MtmsrA and MtmsrB) as part of the repair response to the host-induced oxidative stress. The absence of these enzymes makes Mycobacteria prone to increased susceptibility to cell death, pointing them out as potential therapeutic targets. This study provides a detailed characterization of the catalytic mechanism of MtmsrA using a comprehensive approach, including experimental techniques and theoretical methodologies. Confirming a ping-pong type enzymatic mechanism, we elucidate the catalytic parameters for sulfoxide and thioredoxin substrates (kcat/KM = 2656 ± 525 M-1 s-1 and 1.7 ± 0.8 × 106 M-1 s-1, respectively). Notably, the entropic nature of the activation process thermodynamics, representing ∼85% of the activation free energy at room temperature, is underscored. Furthermore, the current study questions the plausibility of a sulfurane intermediate, which may be a transition-state-like structure, suggesting the involvement of a conserved histidine residue as an acid-base catalyst in the MetSO reduction mechanism. This mechanistic insight not only advances our understanding of Mt antioxidant enzymes but also holds implications for future drug discovery and biotechnological applications.

Pan-Genome-Wide Identification and Transcriptome-Wide Analysis of ZIP Genes in Cucumber

The ZRT/IRT-like proteins (ZIPs) play critical roles in the absorption, transport, and intracellular balance of metal ions essential for various physiological processes in plants. However, little is known about the pan-genomic characteristics and properties of ZIP genes in cucumber (Cucumis sativus L.). In this study, we identified 10 CsZIP genes from the pan-genome of 13 C. sativus accessions. Among them, only CsZIP10 showed no variation in protein sequence length. We analyzed the gene structure, conserved domains, promoter cis-elements, and phylogenetic relationships of these 10 CsZIP genes derived from “9930”. Based on phylogenetic analysis, the CsZIP genes were classified into three branches. Amino acid sequence comparison revealed the presence of conserved histidine residues in the ZIP proteins. Analysis of promoter cis-elements showed that most promoters contained elements responsive to plant hormones. Expression profiling in different tissues showed that most CsZIP genes were expressed at relatively low levels in C. sativus leaves, stems, and tendrils, and CsZIP7 and CsZIP10 were specifically expressed in roots, indicating their potential involvement in the absorption and transport of metal ions. Transcriptomic data indicated that these 10 ZIP genes displayed responses to both downy mildew and powdery mildew, and CsZIP1 was significantly downregulated after both salt and heat treatments. In conclusion, this study deepens our understanding of the ZIP gene family and enhances our knowledge of the biological functions of CsZIP genes in C. sativus.

Overview on tyrosinases: Genetics, molecular biology, phylogenetic relationship.

Tyrosinases (TYRs) are enzymes found in various organisms that are crucial for melanin biosynthesis, coloration, and UV protection. They play vital roles in insect cuticle sclerotization, mollusk shell formation, fungal and bacterial pigmentation, biofilm formation, and virulence. Structurally, TYRs feature copper-binding sites that are essential for catalytic activity, facilitating substrate oxidation via interactions with conserved histidine residues. TYRs exhibit diversity across animals, plants, fungi, mollusks, and bacteria, reflecting their roles and function. Eukaryotic TYRs undergo post-translational modifications, such as glycosylation, which affect protein folding and activity. Bacterial TYRs are categorized into five types based on their structural variation, domain organization and enzymatic properties, showing versatility across bacterial species. Moreover, bacterial TYRs, akin to fungal TYRs, have been implicated in the synthesis of secondary metabolites with antimicrobial properties. TYRs share significant sequence homology with hemocyanins, oxygen-carrier proteins in mollusks and arthropods, highlighting their evolutionary relationships. The evolution of TYRs underscores the dynamic nature of these enzymes and reflects adaptive strategies across diverse taxa.

Role of prophenoloxidase 1 from the beetle Octodonta nipae in melanized encapsulation of a wasp egg

Exploring the function of the host immune system can help to understand the host-parasitoid interaction. Prophenoloxidase (PPO) is crucial in defensive melanization during the encapsulation of wasp eggs. However, the existence of multiple PPO sequences increases the difficulty of exploring the specific functions of individual PPOs. We previously identified three PPOs in the nipa palm hispid beetle, Octodonta nipae. Our current work showed that OnPPO1 and OnPPO2 possessed the typical characteristics of the type III copper family, but OnPPO3 lacked the conserved histidine residues, and its active sites were substituted with Gln. OnPPOs showed the highest expression in hemocytes, but OnPPO3 presented extremely low abundance compared with that of OnPPO1 and OnPPO2, and only OnPPO1 showed a quick response after wasp infection. OnPPO1 knockdown decreased the encapsulation index and inhibited melanization, whereas silencing of OnPPO3 appeared to have no adverse effect on encapsulation and melanization, and silencing of OnPPO2 presented low RNAi efficiency. Moreover, the cleavage of recombinant OnPPO1 produced a 62 kDa fragment with high PO activity. OnPPO1 could be produced by oenocytoids, granulocytes and plasmatocytes, and was distributed around wasp eggs during capsule formation. Overall, our results indicate that proteolytic cleavage of OnPPO1 plays key roles in the melanized encapsulation of wasp eggs. This finding illuminates the mechanism of PPO activation in this invasive beetle and provides guidance for its biological control.

Kinetic and structural details of urease inactivation by thiuram disulphides

This paper reports on the molecular details of the reactivity of urease, a nickel-dependent enzyme that catalyses the last step of organic nitrogen mineralization, with thiuram disulphides, a class of molecules known to inactivate the enzyme with high efficacy but for which the mechanism of action had not been yet established. IC50 values of tetramethylthiuram disulphide (TMTD or Thiram) and tetraethylthiuram disulphide (TETD or Disulfiram) in the low micromolar range were determined for plant and bacterial ureases. The X-ray crystal structure of Sporosarcina pasteurii urease inactivated by Thiram, determined at 1.68 Å resolution, revealed the presence of a covalent modification of the catalytically essential cysteine residue. This is located on the flexible flap that modulates the size of the active site channel and cavity. Formation of a Cys-S-S-C(S)-N(CH3)2 functionality responsible for enzyme inactivation was observed. Quantum-mechanical calculations carried out to rationalise the large reactivity of the active site cysteine support the view that a conserved histidine residue, adjacent to the cysteine in the active site flap, modulates the charge and electron density along the thiol SH bond by shifting electrons towards the sulphur atom and rendering the thiol proton more reactive. We speculate that this proton could be transferred to the nickel-coordinated urea amide group to yield a molecule of ammonia from the generated Curea-NH3+ functionality during catalysis.

Integrative Analysis of Metabolome and Transcriptome Revealed Lutein Metabolism Contributed to Yellow Flower Formation in Prunus mume.

Prunus mume is a famous ornamental woody tree with colorful flowers. P. mume with yellow flowers is one of the most precious varieties. Regretfully, metabolites and regulatory mechanisms of yellow flowers in P. mume are still unclear. This hinders innovation of flower color breeding in P. mume. To elucidate the metabolic components and molecular mechanisms of yellow flowers, we analyzed transcriptome and metabolome between 'HJH' with yellow flowers and 'ZLE' with white flowers. Comparing the metabolome of the two varieties, we determined that carotenoids made contributions to the yellow flowers rather than flavonoids. Lutein was the key differential metabolite to cause yellow coloration of 'HJH'. Transcriptome analysis revealed significant differences in the expression of carotenoid cleavage dioxygenase (CCD) between the two varieties. Specifically, the expression level of PmCCD4 was higher in 'ZLE' than that in 'HJH'. Moreover, we identified six major transcription factors that probably regulated PmCCD4 to affect lutein accumulation. We speculated that carotenoid cleavage genes might be closely related to the yellow flower phenotype in P. mume. Further, the coding sequence of PmCCD4 has been cloned from the 'HJH' petals, and bioinformatics analysis revealed that PmCCD4 possessed conserved histidine residues, ensuring its enzymatic activity. PmCCD4 was closely related to PpCCD4, with a homology of 98.16%. Instantaneous transformation analysis in petal protoplasts of P. mume revealed PmCCD4 localization in the plastid. The overexpression of PmCCD4 significantly reduced the carotenoid content in tobacco plants, especially the lutein content, indicating that lutein might be the primary substrate for PmCCD4. We speculated that PmCCD4 might be involved in the cleavage of lutein in plastids, thereby affecting the formation of yellow flowers in P. mume. This work could establish a material and molecular basis of molecular breeding in P. mume for improving the flower color.

Study on the Synthesis of Theaflavin-3,3′-Digallate Catalyzed by Escherichia coli Expressing Tea Tree Polyphenol Oxidase Isozymes and Its Enzymatic Solution

Polyphenol oxidase and its isoenzymes are crucial enzymes in the tea tree that catalyze the synthesis of theaflavins. In this study, tea tree polyphenol oxidase was used as the research object, and various protein sequence treatments, such as TrxA fusion tag + N-terminal truncation, were tested for prokaryotic expression through the Escherichia coli expression system. Comparative analyses were conducted on the activities of the different recombinant enzyme proteins on the substrates of tea polyphenol fractions. Additionally, the enzyme with the highest catalytic efficiency on the TFDG substrate was immobilized using polyethylene glycol to investigate the yield of its synthesis of TFDG. Our results demonstrated that after N-terminal truncation and TrxA fusion expression, CsPPO1, CsPPO2, CsPPO3, and CsPPO4 were mostly expressed in the form of inclusion bodies in the cell and exhibited varying degrees of enhancement in substrate activity. Specifically, CsPPO1 exhibited significantly increased activity in EC and ECG, CsPPO2 showed enhanced activity towards ECG and EGCG, and CsPPO2 displayed the highest activity toward TFDG substrates. Homology modeling structural analysis of the polyphenol oxidase isozymes revealed that the active centers of CsPPO1, CsPPO2, and CsPPO3 consisted of double copper ion center structures, while the conserved histidine residues surrounding the active centers formed different catalytic activity centers in different structures. Furthermore, polyethylene glycol immobilization significantly increased the activity recovery of the CsPPO2 enzyme to 74.41%. In summary, our study elucidated that tea tree polyphenol oxidase is expressed as inclusion bodies in prokaryotic expression, and the activity of the recombinant enzyme towards substrates could be enhanced through N-terminal truncation and TrxA fusion expression. Moreover, immobilization treatment of the CsPPO2 enzyme greatly improved enzyme efficiency. These findings offer an important enzymatic basis and theoretical support for the synthesis of theaflavins.

Structural insight into the human SID1 transmembrane family member 2 reveals its lipid hydrolytic activity

The systemic RNAi-defective (SID) transmembrane family member 2 (SIDT2) is a putative nucleic acid channel or transporter that plays essential roles in nucleic acid transport and lipid metabolism. Here, we report the cryo-electron microscopy (EM) structures of human SIDT2, which forms a tightly packed dimer with extensive interactions mediated by two previously uncharacterized extracellular/luminal β-strand-rich domains and the unique transmembrane domain (TMD). The TMD of each SIDT2 protomer contains eleven transmembrane helices (TMs), and no discernible nucleic acid conduction pathway has been identified within the TMD, suggesting that it may act as a transporter. Intriguingly, TM3-6 and TM9-11 form a large cavity with a putative catalytic zinc atom coordinated by three conserved histidine residues and one aspartate residue lying approximately 6 Å from the extracellular/luminal surface of the membrane. Notably, SIDT2 can hydrolyze C18 ceramide into sphingosine and fatty acid with a slow rate. The information presented advances the understanding of the structure-function relationships in the SID1 family proteins.

Free ferrous ions sustain activity of mammalian stearoyl-CoA desaturase-1

Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid in a reaction catalyzed by a diiron center. The diiron center is well-coordinated by conserved histidine residues and is thought to remain with the enzyme. However, we find here that SCD1 progressively loses its activity during catalysis and becomes fully inactive after about nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center and that the addition of free ferrous ions (Fe2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe2+ is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe2+ in cells could regulate SCD1 activity and hence lipid metabolism.

Functional and structural diversification of incomplete phosphotransferase system in cellulose-degrading clostridia.

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.

Molecular docking analysis of juglone with parvulin-type PPiase PrsA from Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that causes variety of infections range from mild skin diseases to life-threatening sepsis. It is also notorious for acquiring resistance to numerous antibiotics. Parvulin-type peptidyl-prolyl cis-trans isomerase (PPiase) domain containing PrsA protein is considered as an essential folding factor for secreted proteins of Gram-positive bacteria. Therefore, it is considered as a potential target for anti-staphylococcal drug discovery. Juglone, plant-derived 1,4-naphthoquinone, shows confirmed antitumor and antibacterial activities. Destruction of bacterial biofilm, inhibition of enzyme expression, degradation of nucleic acids, and other pathways are likely the major possible mechanisms for Staphylococcus aureus inactivation by juglone. Selective inhibition of parvulin type PPiase by juglone has been proven biochemically. However, detail structural information of parvulin-juglone interaction and mechanism of enzymatic inhibition till unexplored. Past hypothesis on inactivation of parvulin type PPiase due to covalent attachment of juglone molecules to its cysteine residues is not acceptable for the S. aureus PrsA parvulin domain as that lacks cysteine. Docking studies showed that juglone binds to the active site residues of S. aureus PrsA parvulin domain involved in enzymatic reaction. Active site conserved histidine residue of parvulin may be involved in juglone interaction as it was found to be the common interactive residue in majority of docking complexes. Data shows Juglone possibly inhibits parvulin type PPiase through competitive inhibition mechanism. Subtle differences of juglone interactions with other orthologous parvulin domains will help to develop semisynthetic drug with higher specificity against S. aureus.

Bacterial Two Component Systems: Overexpression and Purification: In Vitro and In Vivo Inhibitor Screens.

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy, such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses upon cell wall or cell membrane damage. However, (i) activity assays using truncated kinase proteins, that is, the cytoplasmic domains containing the conserved histidine residue for phosphorylation, have been shown to produce artifacts, and (ii) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from Gram-positive bacteria that do not harbor more than two trans-membrane domains in an Escherichia coli host. This guide also presents in vitro and in vivo phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases, either using a traditional radioactively labeled ATP assay to quantify histidine kinase phosphorylation or Phos-tag acrylamide gel electrophoresis to examine histidine kinase phosphorylation through mobility shift in the polyacrylamide gel. In addition, we describe the use of Phos-tag combined with a western blot approach to visualize the phosphorylation of a response regulator in vivo.

The role of sensory kinase proteins in two-component signal transduction.

Two-component systems (TCSs) are modular signaling circuits that regulate diverse aspects of microbial physiology in response to environmental cues. These molecular circuits comprise a sensor histidine kinase (HK) protein that contains a conserved histidine residue, and an effector response regulator (RR) protein with a conserved aspartate residue. HKs play a major role in bacterial signaling, since they perceive specific stimuli, transmit the message across the cytoplasmic membrane, and catalyze their own phosphorylation, and the trans-phosphorylation and dephosphorylation of their cognate response regulator. The molecular mechanisms by which HKs co-ordinate these functions have been extensively analyzed by genetic, biochemical, and structural approaches. Here, we describe the most common modular architectures found in bacterial HKs, and address the operation mode of the individual functional domains. Finally, we discuss the use of these signaling proteins as drug targets or as sensing devices in whole-cell biosensors with medical and biotechnological applications.

A Computer Simulation Insight into the Formation of Apocarotenoids: Study of the Carotenoid Oxygenases BCO1 and BCO2 and Their Interaction with Putative Substrates.

Carotenoids are isoprenoid pigments, and sources of vitamin A in humans. The first metabolic pathway for their synthesis is mediated by the enzymes β,β-carotene-15,15'-dioxygenase (BCO1) and β,β-carotene-9',10'-dioxygenase (BCO2), which cleave carotenoids into smaller compounds, called apocarotenoids. The objective of this study is to gain insight into the interaction of BCO1 and BCO2 with carotenoids, adding structural diversity and importance in the agro-food and/or health sectors. Homology modeling of BCO1 and BCO2, and the molecular dynamics of complexes with all carotenoids were performed. Interaction energy and structures were analyzed. For both enzymes, the general structure is conserved with a seven beta-sheet structure, and the β-carotene is positioned at an optimal distance from the catalytic center. Fe2+ forms in an octahedral coordination sphere with four perfectly conserved histidine residues. BCO1 finds stability in a structure in which the β-carotene is positioned ready for enzymatic catalysis at the 15-15' bond, and BCO2 in positioning the bond to be cleaved (C9-C10) close to the active site. In BCO1 the carotenoids interact with only seven residues with aromatic rings, while the interaction of BCO2 is much more varied in terms of the type of interaction, with more residues of different chemical natures.

Structural analysis of the pseudaminic acid synthase PseI from Campylobacter jejuni

Campylobacter jejuni PseI is a pseudaminic acid synthase that condenses the 2,4-diacetamido-2,4,6-trideoxy-l-altrose sugar (6-deoxy AltdiNAc) and phosphoenolpyruvate to generate pseudaminic acid, a sialic acid-like 9-carbon backbone α-keto sugar. Pseudaminic acid is conjugated to cell surface proteins and lipids and plays a key role in the mobility and virulence of C. jejuni and other pathogenic bacteria. To provide insights into the catalytic mechanism of PseI, we performed a structural study on PseI. PseI forms a two-domain structure and assembles into a domain-swapped homodimer. The PseI dimer has two cavities, each of which accommodates a metal ion using conserved histidine residues. A comparative analysis of structures and sequences suggests that the cavity of PseI functions as an active site that binds the 6-deoxy AltdiNAc and phosphoenolpyruvate substrates and mediates their condensation. Furthermore, we propose the substrate binding-induced structural rearrangement of PseI and predict 6-deoxy AltdiNAc recognition residues that are specific to PseI.