Detailed structural characterization of five water-insoluble α-glucans produced by glucansucrases from Streptococcus spp.

Water-insoluble α-glucans synthesized from sucrose by glucansucrases from Streptococcus spp. are essential in dental plaque and caries formation. Because limited information is available on the fine structure of these biopolymers, we analyzed the structures of unmodified glucans produced by five recombinant Streptococcus (S.) mutans DSM 20523 and S. salivarius DSM 20560 glucansucrases in detail. A combination of methylation analysis, endo-dextranase and endo-mutanase hydrolyses, and HPSEC-RI was used. Furthermore, crystal-like regions were analyzed by using XRD and 13C MAS NMR spectroscopy. Our results showed that the glucan structures were highly diverse: Two glucans with 1,3- and 1,6-linkages were characterized in detail besides an almost exclusively 1,3-linked and a linear 1,6-linked glucan. Furthermore, one glucan contained 1,3-, 1,4-, and 1,6-linkages and thus had an unusual, not yet described structure. It was demonstrated that the glucans had a varying structural architecture by using partial enzymatic hydrolyses. Furthermore, crystal-like regions formed by 1,3-glucopyranose units were observed for the two 1,3- and 1,6-linked glucans and the linear 1,3-linked glucan. 1,6-linked regions were mobile and not involved in the crystal-like areas. Altogether, our results broaden the knowledge of the structure of water-insoluble α-glucans from Streptococcus spp.

Biochemical properties of a Flavobacterium johnsoniae dextranase and its biotechnological potential for Streptococcus mutans biofilm degradation.

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.

Tetracyclic homoisoflavanoid (+)-brazilin: a natural product inhibits c-di-AMP-producing enzyme and Streptococcus mutans biofilms.

The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments. There is a growing need for novel therapeutics that selectively inhibit S. mutans biofilms while preserving the normal oral microenvironment. Previous studies have shown that increased levels of cyclic di-AMP, an important secondary messenger synthesized by diadenylate cyclase (DAC), favored biofilm formation in S. mutans. Thus, targeting S. mutans DAC is a novel strategy to inhibit S. mutans biofilms. We screened a small NCI library of natural products using a fluorescence detection assay. (+)-Brazilin, a tetracyclic homoisoflavanoid found in the heartwood of Caesalpinia sappan, was identified as one of the 11 "hits," with the greatest reduction (>99%) in fluorescence at 100 µM. The smDAC inhibitory profiles of the 11 "hits" established by a quantitative high-performance liquid chromatography assay revealed that (+)-brazilin had the most enzymatic inhibitory activity (87% at 100 µM) and was further studied to determine its half maximal inhibitory concentration (IC50 = 25.1 ± 0.98 µM). (+)-Brazilin non-competitively inhibits smDAC's enzymatic activity (Ki = 140.0 ± 27.13 µM), as determined by a steady-state Michaelis-Menten kinetics assay. In addition, (+)-brazilin's binding profile with smDAC (Kd = 11.87 µM) was illustrated by a tyrosine intrinsic fluorescence quenching assay. Furthermore, at low micromolar concentrations, (+)-brazilin selectively inhibited the biofilm of S. mutans (IC50 = 21.0 ± 0.60 µM) and other oral bacteria. S. mutans biofilms were inhibited by a factor of 105 in colony-forming units when treated with 50 µM (+)-brazilin. In addition, a significant dose-dependent reduction in extracellular DNA and glucan levels was evident by fluorescence microscopy imaging of S. mutans biofilms exposed to different concentrations of (+)-brazilin. Furthermore, colonization of S. mutans on a representative model of enamel using suspended hydroxyapatite discs showed a >90% reduction with 50 µM (+)-brazilin. In summary, we have identified a drug-like natural product inhibitor of S. mutans biofilm that not only binds to smDAC but can also inhibit the function of smDAC. (+)-Brazilin could be a good candidate for further development as a potent therapeutic for the prevention and treatment of dental caries.IMPORTANCEThis study represents a significant advancement in our understanding of potential therapeutic options for combating cariogenic biofilms produced by Streptococcus mutans. The research delves into the use of (+)-brazilin, a natural product, as a potent inhibitor of Streptococcus mutans' diadenylate cyclase (smDAC), an enzyme crucial in the formation of biofilms. The study establishes (+)-brazilin as a non-competitive inhibitor of smDAC while providing initial insights into its binding mechanism. What makes this finding even more promising is that (+)-brazilin does not limit its inhibitory effects to S. mutans alone. Instead, it demonstrates efficacy in hindering biofilms in other oral bacteria as well. The broader spectrum of anti-biofilm activity suggests that (+)-brazilin could potentially serve as a versatile tool in a natural product-based treatment for combating a range of conditions caused by resilient biofilms.

Synthesis, Antimicrobial Activities, and Molecular Modeling Studies of Agents for the Sortase A Enzyme.

Sortase A (SrtA) is an attractive target for developing new anti-infective drugs that aim to interfere with essential virulence mechanisms, such as adhesion to host cells and biofilm formation. Herein, twenty hydroxy, nitro, bromo, fluoro, and methoxy substituted chalcone compounds were synthesized, antimicrobial activities and molecular modeling strategies against the SrtA enzyme were investigated. The most active compounds were found to be T2, T4, and T19 against Streptococcus mutans (S. mutans) with MIC values of 1.93, 3.8, 3.94 µg/mL, and docking scores of -6.46, -6.63, -6.73 kcal/mol, respectively. Also, these three active compounds showed better activity than the chlorohexidine (CHX) (MIC value: 4.88 µg/mL, docking score: -6.29 kcal/mol) in both in vitro and in silico. Structural stability and binding free energy analysis of S.mutans SrtA with active compounds were measured by molecular dynamic (MD) simulations throughout 100 nanoseconds (ns) time. It was observed that the stability of the critical interactions between these compounds and the target enzyme was preserved. To prove further, in vivo biological evaluation studies could be conducted for the most promising precursor compounds T2, T4, and T19, and it might open new avenues to the discovery of more potent SrtA inhibitors.

Molecular mechanisms of inhibiting glucosyltransferases for biofilm formation in Streptococcus mutans.

Glucosyltransferases (Gtfs) play critical roles in the etiology and pathogenesis of Streptococcus mutans (S. mutans)- mediated dental caries including early childhood caries. Gtfs enhance the biofilm formation and promotes colonization of cariogenic bacteria by generating biofilm extracellular polysaccharides (EPSs), the key virulence property in the cariogenic process. Therefore, Gtfs have become an appealing target for effective therapeutic interventions that inhibit cariogenic biofilms. Importantly, targeting Gtfs selectively impairs the S. mutans virulence without affecting S. mutans existence or the existence of other species in the oral cavity. Over the past decade, numerous Gtfs inhibitory molecules have been identified, mainly including natural and synthetic compounds and their derivatives, antibodies, and metal ions. These therapeutic agents exert their inhibitory role in inhibiting the expression gtf genes and the activities and secretion of Gtfs enzymes with a wide range of sensitivity and effectiveness. Understanding molecular mechanisms of inhibiting Gtfs will contribute to instructing drug combination strategies, which is more effective for inhibiting Gtfs than one drug or class of drugs. This review highlights our current understanding of Gtfs activities and their potential utility, and discusses challenges and opportunities for future exploration of Gtfs as a therapeutic target.

Protein Interactomes of Streptococcus mutans YidC1 and YidC2 Membrane Protein Insertases Suggest SRP Pathway-Independent- and -Dependent Functions, Respectively.

Virulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. Bacterial cotranslational protein trafficking involves the signal recognition particle (SRP) pathway components Ffh and FtsY, the SecYEG translocon, and YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway and has two yidC paralogs. This study characterized YidC1 and YidC2 interactomes to clarify respective functions alone and in concert with the SRP and/or Sec translocon. Western blots of formaldehyde cross-linked or untreated S. mutans lysates were reacted with anti-Ffh, anti-FtsY, anti-YidC1, or anti-YidC2 antibodies followed by mass spectrometry (MS) analysis of gel-shifted bands. Cross-linked lysates of wild-type and ΔyidC2 strains were reacted with anti-YidC2-coupled Dynabeads, and cocaptured proteins were identified by MS. Last, YidC1 and YidC2 C-terminal tail-captured proteins were subjected to two-dimensional (2D) difference gel electrophoresis and MS analysis. Direct interactions of putative YidC1 and YidC2 binding partners were confirmed by bacterial two-hybrid assay. Our results suggest YidC2 works preferentially with the SRP pathway, while YidC1 is preferred for SRP-independent Sec translocon-mediated translocation. YidC1 and YidC2 autonomous pathways were also apparent. Two-hybrid assay identified interactions between holotranslocon components SecYEG/YajC and YidC1. Both YidC1 and YidC2 interacted with Ffh, FtsY, and chaperones DnaK and RopA. Putative membrane-localized substrates HlyX, LemA, and SMU_591c interacted with both YidC1 and YidC2. Identification of several Rgp proteins in the YidC1 interactome suggested its involvement in bacitracin resistance, which was decreased in ΔyidC1 and SRP-deficient mutants. Collectively, YidC1 and YidC2 interactome analyses has further distinguished these paralogs in the Gram-positive bacterium S. mutansIMPORTANCEStreptococcus mutans is a prevalent oral pathogen and major causative agent of tooth decay. Many proteins that enable this bacterium to thrive in its environmental niche and cause disease are embedded in its cytoplasmic membrane. The machinery that transports proteins into bacterial membranes differs between Gram-negative and Gram-positive organisms, an important difference being the presence of multiple YidC paralogs in Gram-positive bacteria. Characterization of a protein's interactome can help define its physiological role. Herein, we characterized the interactomes of S. mutans YidC1 and YidC2. Results demonstrated substantial overlap between their interactomes but also revealed several differences in their direct protein binding partners. Membrane transport machinery components were identified in the context of a large network of proteins involved in replication, transcription, translation, and cell division/cell shape. This information contributes to our understanding of protein transport in Gram-positive bacteria in general and informs our understanding of S. mutans pathogenesis.

Design, synthesis and evaluation of carbazole derivatives as potential antimicrobial agents.

Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 µg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5-2 µg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure-activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.

Potential Fatty Acid as Antibacterial Agent Against Oral Bacteria of Streptococcus mutans and Streptococcus sanguinis from Basil (Ocimum americanum): In vitro and In silico Studies.

BACKGROUND: Streptococcus mutans and Streptococcus sanguinis are Gram-positive bacteria that cause dental caries. MurA enzyme acts as a catalyst in the formation of peptidoglycan in bacterial cell walls, making it ideal as an antibacterial target. Basil (Ocimum americanum) is an edible plant that is diverse and has been used as a herbal medicine for a long time. It has been reported that basil has a pharmacological effect as well as antibacterial activity. The purpose of this study was to identify antibacterial compounds in O. americanum and analyze their inhibition activity on MurA enzyme. METHODS: Fresh leaves from O. americanum were extracted with n-hexane and purified by a combination of column chromatography on normal and reverse phases together with in vitro bioactivity assay against S. mutans ATCC 25175 and S. sanguinis ATCC 10556, respectively, while in silico molecular docking simulation of lauric acid (1) was conducted using PyRx 0.8. RESULTS: The structure determination of antibacterial compound by spectroscopic methods resulted in an active compound lauric acid (1). The in vitro evaluation of antibacterial activity in compound 1 showed Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of 78.13 and 156.3 ppm and 1250 and 2500 ppm against S. sanguinis and S. mutans, respectively. Further analysis and in silico evaluation determined lauric acid (1) as MurA Enzyme inhibitor. Lauric acid (1) showed a binding affinity of -5.2 Kcal/mol, which was higher than fosfomycin. CONCLUSION: Lauric acid showed the potential as a new natural antibacterial agent through MurA inhibition in bacterial cell wall biosynthesis.

Significance of Individual Domains of ClpL: A Novel Chaperone from Streptococcus mutans.

ClpL is a member of the HSP100 family of AAA+ chaperones that is widely present in Gram-positive but surprisingly absent in Gram-negative bacteria. ClpL is involved in various cellular processes, including stress tolerance response, long-term survival, virulence, and antibiotic resistance. ClpL is poorly characterized, and its molecular mechanisms of chaperone activity are largely unclear. Here, we biochemically characterized the ClpL protein from Streptococcus mutans, a dental pathogen, to understand its biological functions. ClpL harbors five domains: N-domain, two nucleotide binding domains (NBD-1 and NBD-2), M-domain, and C-domain. NBD-1 and NBD-2 contain distinct Walker A and B motifs for ATP binding and hydrolysis, respectively. We found that ClpL predominantly exists as a trimer in solution; however, upon ATP binding, it rapidly forms a hexameric structure. To study structure-function activity, we constructed several substitution and deletion mutants. We found that mutations in the Walker A and B motifs interfered with ATP hydrolysis and oligomerization. Similarly, deletions of N-, M-, and C-domains abolished both ATPase activity and oligomerization. Because we previously found that ClpL acts as a chaperone, we analyzed the chaperone activity. Surprisingly, we found that the NBD-2 mutants did not display any chaperone activity, indicating that ATP binding and hydrolysis by NBD-2 are essential for the chaperone. However, NBD-1 mutants showed chaperone activities, but the activities were variable depending on the nature of the mutations. Our results indicate that unlike other HSP100 family chaperones, ClpL is a novel chaperone that does not require any additional secondary chaperones for its activity.

Design, green one-pot synthesis and molecular docking study of novel N,N-bis(cyanoacetyl)hydrazines and bis-coumarins as effective inhibitors of DNA gyrase and topoisomerase IV.

A novel, quick, environmentally safe, and one-pot synthesis of a series of N,N-bis(cyanoacetyl)hydrazine derivatives, bis-imino-2H-chromenes and bis-2-oxo-2H-chromene derivatives have been designed. Some selected newly synthesized compounds were investigated in vitro for their antibacterial activity. Compound 5j is the most toxic compound against Staphylococcus aureus with activity index 171%, followed by compound 15b with activity index 136% compared to standard drug ampicillin. Moreover, compound 15a is the most toxic compound against Escherichia coli with activity index 111% compared to standard drug gentamicin. Minimum inhibitory concentration (MIC) was carried out for compounds with high antibacterial activity. Compound 5j has good MIC (7.8 µg/ml) against Staphylococcus aureus while 15a has good MIC (31.25 µg/ml) against Streptococcus mutans which is better than MIC of the standard drug ampicillin (MIC = 62.5 µg/ml). Compounds 5j, 5k, 15a, 15b and 15e which have good MIC values were introduced to enzyme assay against DNA gyrase and topoisomerase IV. The results showed that compound 15a can strongly inhibit DNA gyrase and topoisomerase IV (IC50 = 27.30 and 25.52 µM respectively), compared to methotrexate as the standard drug (IC50 = 29.01 and 23.55 µM respectively). Structure-activity relationships were also discussed based on the biological and docking simulation results.

Antimicrobial activity of rhodomyrtone isolated from Rhodomyrtus tomentosa (Aiton) Hassk.

Rhodomyrtone was isolated from the leaves of Rhodomyrtus tomentosa (Aiton) Hassk grown in Vietnam using chromatographic methods. Its chemical structure was confirmed by means of spectroscopic data analysis. The pH drop measurement, enzyme activity assays and fluorescence stain were used to examine rhodomyrtone anticaries activity. It was found that rhodomyrtone suppressed acid production by Streptococcus mutans, a major cariogenic agent in human by inhibiting enzyme activities responsible for acid production and tolerance, including membrane bound enzymes F-ATPase and phosphotransferase system (PTS), as well as glycolysis enzymes glyceraldehyphosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) in cytoplasm with the IC50 values of 24 µM, 19 µM, 23 µM and 28 µM, respectively. Moreover, 50 µM rhodomyrtone reduced biofilm biomass formed by S. mutans up to 59% (p < 0.05). Fluorescent images indicated that cells on the biofilms were significantly killed. Thus, rhodomyrtone is a new and potential anticaries agent against S. mutans.

Quantitative Proteomics Uncovers the Interaction between a Virulence Factor and Mutanobactin Synthetases in Streptococcus mutans.

Streptococcus mutans, the primary etiological agent of tooth decay, has developed multiple adhesion and virulence factors which enable it to colonize and compete with other bacteria. The putative glycosyltransferase SMU_833 is important for the virulence of S. mutans by altering the biofilm matrix composition and cariogenicity. In this study, we further characterized the smu_833 mutant by evaluating its effects on bacterial fitness. Loss of SMU_833 led to extracellular DNA-dependent bacterial aggregation. In addition, the mutant was more susceptible to oxidative stress and less competitive against H2O2 producing oral streptococci. Quantitative proteomics analysis revealed that SMU_833 deficiency resulted in the significant downregulation of 10 proteins encoded by a biosynthetic gene cluster responsible for the production of mutanobactin, a compound produced by S. mutans which helps it survive oxidative stress. Tandem affinity purification demonstrated that SMU_833 interacts with the synthetic enzymes responsible for the production of mutanobactin. Similar to the smu_833 mutant, the deletion of the mutanobactin gene cluster rendered the mutant less competitive against H2O2-producing streptococci. Our studies revealed a new link between SMU_833 virulence and mutanobactin, suggesting that SMU_833 represents a new virulent target that can be used to develop potential anticaries therapeutics.IMPORTANCEStreptococcus mutans is the major bacterium associated with dental caries. In order to thrive on the highly populated tooth surface and cause disease, S. mutans must be able to protect itself from hydrogen peroxide-producing commensal bacteria and compete effectively against the neighboring microbes. S. mutans produces mutacins, small antimicrobial peptides which help control the population of competing bacterial species. In addition, S. mutans produces a peptide called mutanobactin, which offers S. mutans protection against oxidative stress. Here, we uncover a new link between the putative glycosyltransferase SMU_833 and the mutanobactin-synthesizing protein complex through quantitative proteomic analysis and a tandem-affinity protein purification scheme. Furthermore, we show that SMU_833 mediates bacterial sensitivity to oxidative stress and bacterial ability to compete with commensal streptococci. This study has revealed a previously unknown association between SMU_833 and mutanobactin and demonstrated the importance of SMU_833 in the fitness of S. mutans.

Design and study of anticaries effect of different medicinal plants against S.mutans glucosyltransferase.

BACKGROUND: The present study was aimed to evaluate the molecular level anticaries effect of different medicinal plants against Streptococcus mutans (S.mutans) glucosyltransferases (gtf). METHODS: A total of six natural sources named as Terminalia chebula (T.chebula), Psidium guajava (P.guajava), Azadirachta indica (A.indica) and Pongamia pinnata (P.pinnata); two essential oils, clove (Syzygium aromaticum) and peppermint oil (Mentha piperita) were selected as test samples. Hydroalcoholic plant extracts and essential oils were examined for their inhibitory potential on gtf isolated from S.mutans. Polyherbal mouth wash was prepared and its effect on gtf activity was compared with commercial chlorhexidine mouth wash (5%w/v). Enzyme kinetic study was carried out in order to explore the molecular mechanism of enzyme action. RESULTS: Out of six natural sources tested, A.indica has shown maximum inhibitory effect of 91.647% on gtf and T.chebula has shown IC50 of 1.091 mg/ml which is significant when compared to standard chlorhexidine. From the final result of kinetic analysis it was found that T.chebula, P.guajava and P.pinnata have show uncompetitive inhibition where as A.indica has shown non-competitive inhibition. Surprisingly, both essential oils have shown allosteric inhibition (sigmoidal response). The polyherbal moutwash has shown significant inhibitory potential on gtf (95.936%) when compared to commercial chlorhexidine mouthwash (p < 0.05). CONCLUSION: All the tested samples have shown considerable gtf inhibitory action. Moreover polyherbal mouth wash has shown promising noncompetitive inhibitory activity against gtf and it could be the future formulation to combat dental caries.

Alginate-pectin co-encapsulation of dextransucrase and dextranase for oligosaccharide production from sucrose feedstocks.

The genes for dextransucrase and dextranase were cloned from the genomic regions of Leuconostoc mesenteroides MTCC 10508 and Streptococcus mutans MTCC 497, respectively. Heterologous expression of genes was performed in Escherichia coli. The purified enzyme fractions were entrapped in the alginate-pectin beads. A high immobilization yield of dextransucrase (~ 96%), and dextranase (~ 85%) was achieved. Alginate-pectin immobilization did not affect the optimum temperature and pH of the enzymes; rather, the thermal tolerance and storage stability of the enzymes was improved. The repetitive batch experiments suggested substantially good operational stability of the co-immobilized enzyme system. The synergistic catalytic reactions of alginate-pectin co-entrapped enzyme system were able to produce 7-10 g L-1 oligosaccharides of a high degree of polymerization (DP 3-9) from sucrose (~ 20 g L-1) containing feedstocks, e.g., table sugar and cane molasses. The alginate-pectin-based co-immobilized enzyme system is a useful catalytic tool to bioprocess the agro-industrial bio-resource for the production of prebiotic biomolecules.

Crystal structure of the 65-kilodalton amino-terminal fragment of DNA topoisomerase I from the gram-positive model organism Streptococcus mutans.

Herein we report the first structure of topoisomerase I determined from the gram-positive bacterium, S. mutans. Bacterial topoisomerase I is an ATP-independent type 1A topoisomerase that uses the inherent torsional strain within hyper-negatively supercoiled DNA as an energy source for its critical function of DNA relaxation. Interest in the enzyme has gained momentum as it has proven to be essential in various bacterial organisms. In order to aid in further biochemical characterization, the apo 65-kDa amino-terminal fragment of DNA topoisomerase I from the gram-positive model organism Streptococcus mutans was crystalized and a three-dimensional structure was determined to 2.06 Šresolution via x-ray crystallography. The overall structure illustrates the four classic major domains that create the traditional topoisomerase I "lock" formation comprised of a sizable toroidal aperture atop what is considered to be a highly dynamic body. A catalytic tyrosine residue resides at the interface between two domains and is known to form a 5' phosphotyrosine DNA-enzyme intermediate during transient single-stranded cleavage required for enzymatic relaxation of hyper negative DNA supercoils. Surrounding the catalytic tyrosine residue is the remainder of the highly conserved active site. Within 5 Šfrom the catalytic center, only one dissimilar residue is observed between topoisomerase I from S. mutans and the gram-negative model organism E. coli. Immediately adjacent to the conserved active site, however, S. mutans topoisomerase I displays a somewhat unique nine residue loop extension not present in any bacterial topoisomerase I structures previously determined other than that of an extremophile.

The role of conserved arginine in the GH70 family: a computational study of the structural features and their implications on the catalytic mechanism of GTF-SI from Streptoccocus mutans.

In this work, molecular dynamics and QM/MM calculations were employed to examine the structural and catalytic features of the retaining glucosyltransferase GTF-SI from the GH70 family, which participates in the process of caries formation. Our goal was to obtain a deeper understanding of the role of R475 in the mechanism of sucrose breakage. This residue is highly conserved in the GH70 family and so far there has been no evidence that shows what could be the role of this residue in the catalysis performed by GTF-SI. In order to understand the structural role of R475 in the native enzyme, we built full enzyme models of the wild type and the mutants R475A and R475Q. These models were addressed by means of molecular dynamics simulations, which allowed the assessment of the dynamical effect of the R475 mutation on the active site. Then, representative structures were chosen for each one of the mutant models and QM/MM calculations were carried out to unravel the catalytic role of R475. Our results show that the R475 mutation increases the flexibility of the enzyme, which triggers the entrance of water molecules in the active site. In addition, QM/MM calculations indicate that R475 is able to provide a great stabilization to the carboxylate moiety of the acid/base E515, which is an essential characteristic favoring the proton transfer process that promotes the glycosidic bond breakage of sucrose.

Amino Acid Residues Recognizing Isomeric Glutamate Substrates in UDP- N-acetylmuramic acid-l-alanine-glutamate Synthetases.

We recently revealed that a previously unknown pathway for peptidoglycan biosynthesis operates in some microorganisms, including Xanthomonas oryzae. It involves two enzymes, MurD2 and MurL, which catalyze the ligation of l-glutamate (l-Glu) to UDP- N-acetylmuramic acid-l-alanine and the epimerization of the terminal l-Glu of the product, respectively. MurD2 of X. oryzae possesses a 26% identity with MurD of Escherichia coli (MurDec), which ligates d-Glu to UDP- N-acetylmuramic acid-l-alanine. To understand how X. oryzae MurD2 recognizes the isomer substrate, we estimated its structure based on that of MurDec during docking simulations. Several amino acid residues, which may be responsible for l-Glu recognition, were replaced with their corresponding amino acid residues in MurDec. Consequently, we obtained a mutated MurD2 enzyme that contained two amino acid substitutions and accepted only d-Glu as the substrate. We next tried to convert the substrate specificity of MurDec using the same strategy, but the mutant enzyme still accepted only d-Glu. Then, MurD of Streptococcus mutans (MurDsm), which possesses the key amino acid residue for l-Glu recognition identified in MurD2, was used for random screenings of mutant enzymes accepting l-Glu. We obtained a mutated MurDsm that had one amino acid substitution and slightly accepted l-Glu. A mutated MurDec possessing the corresponding one amino acid substitution also accepted l-Glu. Thus, we revealed that a few amino acid residues in MurD/MurD2 might control the acceptability of substrates with different stereochemistries.

Proton-pumping F-ATPase plays an important role in Streptococcus mutans under acidic conditions.

Streptococcus mutans, a bacterium mainly inhabiting the tooth surface, is a major pathogen of dental caries. The bacterium metabolizes sugars to produce acids, resulting in an acidic microenvironment in the dental plaque. Hence, S. mutans should possess a mechanism for surviving under acidic conditions. In the current study, we report the effects of inhibitors of Escherichia coli proton-pumping F-type ATPase (F-ATPase) on the activity of S. mutans enzyme, and the growth and survival of S. mutans under acidic conditions. Piceatannol, curcumin, and demethoxycurcumin strongly reduced the ATPase activity of S. mutans F-ATPase. Interestingly, these compounds inhibited the growth of S. mutans at pH 5.3 but not at pH 7.3. They also significantly reduced the colony-forming ability of S. mutans after incubation at pH 4.3, while showing essentially no effect at pH 7.3. These observations indicate that S. mutans is highly sensitive to F-ATPase inhibitors under acidic conditions and that F-ATPase plays an important role in acid tolerance of this bacterium.

Ratiometric fluorescent probe for sensing Streptococcus mutans glucosyltransferase, a key factor in the formation of dental caries.

We report on a naphthalimide ratiometric fluorescent probe for the real-time sensing and imaging of pathogenic bacterial glucosyltransferases, which are associated with the development of dental caries. Using a high-throughput screening method, we identified that several natural polyphenols from green tea were GTFs inhibitors that could eventually lead to suitable oral treatments to prevent the development of dental caries.

Crystal structure of the aromatic-amino-acid aminotransferase from Streptococcus mutans.

Streptococcus mutans, a facultatively aerobic and Gram-positive bacterium, is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. In this study, the aromatic-amino-acid aminotransferase from Streptococcus mutans (SmAroAT) was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The recombinant protein was crystallized using the hanging-drop vapor-diffusion method with PEG 3350 as the primary precipitant. The crystal structure of SmAroAT was solved at 2.2 Šresolution by the molecular-replacement method. Structural analysis indicated that the proteins of the aromatic-amino-acid aminotransferase family have conserved structural elements that might play a role in substrate binding. These results may help in obtaining a better understanding of the catabolism and biosynthesis of aromatic amino acids.