Structure analysis and site-directed mutagenesis of defined key residues and motives for pilus-related sortase C1 in group B Streptococcus

In group B Streptococcus (GBS), 3 structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular-weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies, and in vivo mutagenesis, we performed a broad characterization of GBS sortase C1 of pilus island 2a. The high-resolution X-ray structure of the enzyme revealed that the active site, into the \u3b2-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the \u201clid.\u201d We show that the catalytic triad and the predicted N- and C-terminal transmembrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies, we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild-type protein.\u2014Cozzi, R., Malito, E., Nuccitelli, A., D\u2019Onofrio, M., Martinelli, M., Ferlenghi, I., Grandi, G., Telford, J. L., Maione, D., Rinaudo, C. D. Structure analysis and site-directed mutagenesis of defined key residues and motives for pilus-related sortase C1 in group B Streptococcus

Small-Molecule Activators of Insulin-Degrading Enzyme Discovered through High-Throughput Compound Screening

Background: Hypocatabolism of the amyloid β-protein (Aβ) by insulin-degrading enzyme (IDE) is implicated in the pathogenesis of Alzheimer disease (AD), making pharmacological activation of IDE an attractive therapeutic strategy. However, it has not been established whether the proteolytic activity of IDE can be enhanced by drug-like compounds. Methodology/Principal Findings: Based on the finding that ATP and other nucleotide polyphosphates modulate IDE activity at physiological concentrations, we conducted parallel high-throughput screening campaigns in the absence or presence of ATP and identified two compounds—designated Ia1 and Ia2—that significantly stimulate IDE proteolytic activity. Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE. Unexpectedly, we observed highly synergistic activation effects when the activity of Ia1 or Ia2 was tested in the presence of ATP, a finding that has implications for the mechanisms underlying ATP-mediated activation of IDE. Notably, Ia1 and Ia2 activated the degradation of Aβ by ∼700% and ∼400%, respectively, albeit only when Aβ was presented in a mixture also containing shorter substrates. Conclusions/Significance: This study describes the first examples of synthetic small-molecule activators of IDE, showing that pharmacological activation of this important protease with drug-like compounds is achievable. These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP. Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs

Diversity of a cytokinin dehydrogenase gene in wild and cultivated barley

The cytokinin dehydrogenase gene HvCKX2.1 is the regulatory target for the most abundant heterochromatic small RNAs in drought-stressed barley caryopses. We investigated the diversity of HvCKX2.1 in 228 barley landraces and 216 wild accessions and identified 14 haplotypes, five of these with ten or more members, coding for four different protein variants. The third largest haplotype was abundant in wild accessions (51 members), but absent from the landrace collection. Protein structure predictions indicated that the amino acid substitution specific to haplotype 3 could result in a change in the functional properties of the HvCKX2.1 protein. Haplotypes 1–3 have overlapping geographical distributions in the wild population, but the average rainfall amounts at the collection sites for haplotype 3 plants are significantly higher during November to February compared to the equivalent data for plants of haplotypes 1 and 2. We argue that the likelihood that haplotype 3 plants were excluded from landraces by sampling bias that occurred when the first wild barley plants were taken into cultivation is low, and that it is reasonable to suggest that plants with haplotype 3 are absent from the crop because these plants were less suited to the artificial conditions associated with cultivation. Although the cytokinin signalling pathway influences many aspects of plant development, the identified role of HvCKX2.1 in the drought response raises the possibility that the particular aspect of cultivation that mitigated against haplotype 3 relates in some way to water utilization. Our results therefore highlight the possibility that water utilization properties should be looked on as a possible component of the suite of physiological adaptations accompanying the domestication and subsequent evolution of cultivated barley

The Substrate-Bound Crystal Structure of a Baeyer–Villiger Monooxygenase Exhibits a Criegee-like Conformation

The Baeyer\u2013Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer\u2013Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP+ and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP+ and FAD, to 2.4 \uc5 resolution. This structure shows a drastic rotation of the NADP+ cofactor in comparison to previously reported NADP+-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP+ permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme\u2019s chemospecificity, to a location above the flavin\u2013peroxide adduct where catalysis occurs.Peer reviewed: YesNRC publication: Ye

Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death

This work was supported by Agencia Aragonesa para la Investigación y Desarrollo (ARAID), Ministerio de Economía y Competitividad (CTQ2013-44367-C2-2-P to R.H.-G.) and Diputación General de Aragón (DGA; B89 to R.H.-G.) and the EU Seventh Framework Programme (2007–2013) under BioStruct-X (grant agreement 283570 and BIOSTRUCTX 5186, to R.H.-G.). T.K.S. was supported by the Wellcome Trust grant 093228 and European Community’s Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED).Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite’s growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase.Publisher PDFPeer reviewe

Differential Role of Human Choline Kinase α and β Enzymes in Lipid Metabolism: Implications in Cancer Onset and Treatment

11 pages, 6 figures, 1 table.Background The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of 1phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKα and ChoKβ isoforms, the first one with two different variants of splicing. Recently ChoKα has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKβ in carcinogenesis has been reported. Methodology/Principal Findings Here we compare the in vitro and in vivo properties of ChoKα1 and ChoKβ in lipid metabolism, and their potential role in carcinogenesis. Both ChoKα1 and ChoKβ showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKβ display an ethanolamine kinase role, ChoKα1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKα1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKβ overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKα1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKβ mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKα1 than ChoKβ. Conclusion/Significance This study represents the first evidence of the distinct metabolic role of ChoKα and ChoKβ isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.This work has been supported by grants to JCL from Comunidad de Madrid (GR-SAL-0821-2004), Ministerio de Ciencia e Innovación (SAF2008-03750, RD06/0020/0016), Fundación Mutua Madrileña, and by a grant to ARM from Fundación Mutua Madrileña.Peer reviewe

Supramolecular Organization of the Repetitive Backbone Unit of the Streptococcus pneumoniae Pilus

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 Å resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility