Marek Wojciechowski

The Noncanonical Disulfide Bond as the Important Stabilizing Element of the Immunoglobulin Fold of the Dr Fimbrial DraE Subunit

Fimbrial adhesins of pathogenic bacteria are linear protein associates responsible for binding to the specific host cell receptors. They are assembled via the chaperone/usher pathway conserved in Gram-negative bacteria. These adhesive organelles are characterized by the high resistance to dissociation and unfolding caused by temperature or chemical denaturants. The self-complemented (SC) recombinant subunits of adhesive structures make up the minimal model used to analyze stability phenomena of these organelles. The SC subunits are both highly stabilized thermodynamically and kinetically. They are characterized by a standard free energy of unfolding of 70-80 kJ/mol and a rate constant of unfolding of 10(-17) s(-1) (half-life of unfolding of 10(8) years at 25 degrees C). The DraE subunit of Dr fimbriae is characterized by a disulfide bond that joins the beginning of the A1 strand with the end of the B strand. Such localization is unique and differentiates this protein from other proteins of the Ig-like family. Sequence analysis shows that many protein subunits of adhesive structures possess cysteines that may form a potential disulfide bond homologous to that of DraE. In this paper, we investigate the influence of this noncanonical disulfide bond on the stability of DraE-sc by constructing a DraE-sc-DeltaSS mutant protein (Cys/Ala mutant). This construct unfolds thermally at a T(m) of 65.4 degrees C, more than 20 degrees C lower than that of the native DraE-sc protein, and possesses a different unfolding mechanism. The calculated standard free energy of unfolding of DraE-sc-DeltaSS is equal to 30 +/- 5 kJ/mol. This allows us to suggest that the disulfide bond is an important stabilizing feature of many fimbrial subunits.

Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana

BackgroundIn recent years, there has been an increasing interest in SSBs because they find numerous applications in diverse molecular biology and analytical methods.ResultsWe report the characterization of single-stranded DNA binding proteins (SSBs) from the thermophilic bacteria Thermotoga maritima (Tma SSB) and Thermotoga neapolitana (Tne SSB). They are the smallest known bacterial SSB proteins, consisting of 141 and 142 amino acid residues with a calculated molecular mass of 16.30 and 16.58 kDa, respectively. The similarity between amino acid sequences of these proteins is very high: 90% identity and 95% similarity. Surprisingly, both Tma SSB and Tne SSB possess a quite low sequence similarity to Escherichia coli SSB (36 and 35% identity, 55 and 56% similarity, respectively). They are functional as homotetramers containing one single-stranded DNA binding domain (OB-fold) in each monomer. Agarose mobility assays indicated that the ssDNA-binding site for both proteins is salt independent, and fluorescence spectroscopy resulted in a size of 68 ± 2 nucleotides. The half-lives of Tma SSB and Tne SSB were 10 h and 12 h at 100°C, respectively. When analysed by differential scanning microcalorimetry (DSC) the melting temperature (Tm) was 109.3°C and 112.5°C for Tma SSB and Tne SSB, respectively.ConclusionThe results showed that Tma SSB and Tne SSB are the most thermostable SSB proteins identified to date, offering an attractive alternative to Taq SSB and Tth SSB in molecular biology applications, especially with using high temperature e. g. polymerase chain reaction (PCR).

Pilicides inhibit the FGL chaperone/usher assisted biogenesis of the Dr fimbrial polyadhesin from uropathogenic Escherichia coli

BackgroundThe global spread of bacterial resistance has given rise to a growing interest in new anti-bacterial agents with a new strategy of action. Pilicides are derivatives of ring-fused 2-pyridones which block the formation of the pili/fimbriae crucial to bacterial pathogenesis. They impair by means of a chaperone-usher pathway conserved in the Gram-negative bacteria of adhesive structures biogenesis. Pili/fimbriae of this type belong to two subfamilies, FGS and FGL, which differ in the details of their assembly mechanism. The data published to date have shown that pilicides inhibit biogenesis of type 1 and P pili of the FGS type which are encoded by uropathogenic E. coli strains.ResultsWe evaluated the anti-bacterial activity of literature pilicides as blockers of the assembly of a model example of FGL-type adhesive structures, – the Dr fimbriae encoded by a dra gene cluster of uropathogenic Escherichia coli strains. In comparison to the strain grown without pilicide, the Dr+ bacteria cultivated in the presence of the 3.5 mM concentration of pilicides resulted in a reduction of 75 to 87% in the adherence properties to CHO cells expressing Dr fimbrial DAF receptor protein. Using quantitative assays, we determined the amount of Dr fimbriae in the bacteria cultivated in the presence of 3.5 mM of pilicides to be reduced by 75 to 81%. The inhibition effect of pilicides is concentration dependent, which is a crucial property for their use as potential anti-bacterial agents. The data presented in this article indicate that pilicides in mM concentration effectively inhibit the adherence of Dr+ bacteria to the host cells, – the crucial, initial step in bacterial pathogenesis.ConclusionsStructural analysis of the DraB chaperone clearly showed it to be a model of the FGL subfamily of chaperones. This permits us to conclude that analyzed pilicides in mM concentration are effective inhibitors of the assembly of adhesins belonging to the Dr family, and more speculatively, of other FGL-type adhesive organelles. The presented data and those published so far permit to speculate that based on the conservation of chaperone-usher pathway in Gram-negative bacteria , the pilicides are potential anti-bacterial agents with activity against numerous pathogens, the virulence of which is dependent on the adhesive structures of the chaperone-usher type.

Structural factors affecting affinity of cytotoxic oxathiole-fused chalcones toward tubulin

Synthesis, in vitro cytotoxic activity, and interaction with tubulin of (E)-1-(6-alkoxybenzo[d][1,3]oxathiol-5-yl)-3-phenylprop-2-en-1-one derivatives (2) are described. Some of the compounds demonstrated cytotoxic activity at submicromolar concentrations, and the activity could be related to interaction with tubulin at the colchicine binding site. Interaction of selected derivatives with tubulin was evaluated using molecular modeling, and two different modes of the interaction were identified. The proposed models demonstrate how particular structural fragments participate in binding to the tubulin and explain the importance of the fragments for cytotoxic activity. It was demonstrated that concerning binding to tubulin, the 6-alkoxybenzoxathiole ring can be considered as structural equivalent of trimethoxyphenyl motif of colchicine, podophyllotoxin or combretastatin A4. The observation opened new ways of rational modifications of several groups of tubulin binders.

Homoisocitrate dehydrogenase from Candida albicans: properties, inhibition, and targeting by an antifungal pro-drug

The LYS12 gene from Candida albicans, coding for homoisocitrate dehydrogenase was cloned and expressed as a His-tagged protein in Escherichia coli. The purified gene product catalyzes the Mg(2+)- and K(+)-dependent oxidative decarboxylation of homoisocitrate to α-ketoadipate. The recombinant enzyme demonstrates strict specificity for homoisocitrate. SDS-PAGE of CaHIcDH revealed its molecular mass of 42.6 ± 1 kDa, whereas in size-exclusion chromatography, the enzyme eluted in a single peak corresponding to a molecular mass of 158 ± 3 kDa. Native electrophoresis showed that CaHIcDH may exist as a monomer and as a tetramer and the latter form is favored by homoisocitrate binding. CaHIcDH is an hysteretic enzyme. The K(M) values of the purified His-tagged enzyme for NAD(+) and homoisocitrate were 1.09 mM and 73.7 μM, respectively, and k(cat) was 0.38 s(-1). Kinetic parameters determined for the wild-type CaHIcDH were very similar. The enzyme activity was inhibited by (2R,3S)-3-(p-carboxybenzyl)malate (CBMA), with IC(50) = 3.78 mM. CBMA demonstrated some moderate antifungal activity in minimal media that could be enhanced upon conversion of the enzyme inhibitor into its trimethyl ester derivative (TMCBMA). TMCBMA is the first reported antifungal for which an enzyme of the AAP was identified as a molecular target.

N3-oxoacyl derivatives of L-2,3-diaminopropanoic acid and their peptides; novel inhibitors of glucosamine-6-phosphate synthase.

Abstract Novel inhibitors 1–4 of glucosamine-6-phosphate synthase from Candida albicans have been designed based on acylation of the N3 amino group of L-2,3-diaminopropanoic acid with the corresponding ketoacids. These inhibitors have been shown to alkylate the fungal enzyme in a time-dependent manner. Compound 3 containing trans-β-benzoyl acrylic acid as an acyl residue was found to be the most potent inhibitor in the series. Dipeptides composed of the active inhibitors and norvaline demonstrated potent antifungal activity against selected strains of Candida spp. and Saccharomyces cerevisiae. Their activity was reversed upon addition of N-acetylglucosamine to the medium.

Antifungal Activity of Homoaconitate and Homoisocitrate Analogs

Thirteen structural analogs of two initial intermediates of the l-α-aminoadipate pathway of l-lysine biosynthesis in fungi have been designed and synthesized, including fluoro- and epoxy-derivatives of homoaconitate and homoisocitrate. Some of the obtained compounds exhibited at milimolar range moderate enzyme inhibitory properties against homoaconitase and/or homoisocitrate dehydrogenase of Candida albicans. The structural basis for homoisocitrate dehydrogenase inhibition was revealed by molecular modeling of the enzyme-inhibitor complex. On the other hand, the trimethyl ester forms of some of the novel compounds exhibited antifungal effects. The highest antifungal activity was found for trimethyl trans-homoaconitate, which inhibited growth of some human pathogenic yeasts with minimal inhibitory concentration (MIC) values of 16–32 μg/mL.

Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification.

Rationally designed muteins of Candida albicans glucosamine‐6‐phosphate synthase, an enzyme known as a promising target for antifungal chemotherapy, were constructed, overexpressed in Escherichia coli and purified to near homogeneity. To facilitate and to optimize the purification of the enzyme, three recombinant versions containing internal oligoHis fragments were constructed: (i) by substituting residues 343–348 of the interdomain undecapeptide linker with hexaHis, (ii) by replacing solvent‐exposed residues 655–660 of the isomerase domain with hexaHis, and (iii) by replacing amino acids at positions 568 and 569 with His residues to generate the three‐dimensional hexaHis microdomain in the enzyme quaternary structure. The resulting constructs were effectively purified to near homogeneity by rapid, one‐step immobilized metal‐ion affinity chromatography and demonstrated activity and catalytic properties comparable with that of the wild‐type enzyme. The construct containing the 655–660 hexaHis insert was found to be a homodimeric protein, which is the first reported example of such quaternary structure of glucosamine‐6‐phosphate synthase of eukaryotic origin. Copyright

Synthesis and biological activity of novel ester derivatives of N3-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid containing amide and keto function as inhibitors of glucosamine-6-phosphate synthase

A short series of novel ester derivatives of N(3)-4-metoxyfumaroyl-(S)-2,3-diaminopropanoic acid (FMDP) containing amide or keto functions have been designed and synthesized. Their antifungal activity and inhibitory properties toward fungal glucosamine-6-phosphate synthase has also been evaluated. The obtained compounds 11-13 and 15-17 demonstrated good antifungal activity against Candida albicans. Compounds 11-13 displayed also potent inhibitory activity against fungal glucosamine-6-phosphate synthase, comparable to that of FMDP.

Synthesis, anticandidal activity of N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic amide derivatives – Novel inhibitors of glucosamine-6-phosphate synthase

Novel FMDP amides 4-6 have been synthesized and tested against Candida strains. The anticandidal activity has been confined only to Candida albicans. Anticandidal activity of the tested amides has correlated with their inhibitory activity of glucosamine-6-phosphate synthase in cell free extract from C. albicans.