scholarly journals GlcNAc De-N-Acetylase from the Hyperthermophilic ArchaeonSulfolobus solfataricus

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Roberta Iacono ◽  
Andrea Strazzulli ◽  
Luisa Maurelli ◽  
Nicola Curci ◽  
Angela Casillo ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Recombinant Enzyme ◽  
Mass Spectrometry Analysis ◽  
Genomic Context ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Additional Information ◽  
The One ◽  
Hydrolysis Of

ABSTRACTSulfolobus solfataricusis an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group ofSulfolobales,N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of theN-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood inArchaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening ofS. solfataricusextracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified inArchaea. Mass spectrometry analysis of the purified activity showed a protein encoded by thesso2901gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed inS. solfataricusextracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream ofsso2901(sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism inS. solfataricus.IMPORTANCEIn this study, a preliminary screening of cellular extracts ofS. solfataricusallowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism ofArchaea.

scholarly journals Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii

2018 ◽  
Author(s):  
Rahul Salunke ◽  
Tobias Mourier ◽  
Manidipa Banerjee ◽  
Arnab Pain ◽  
Dhanasekaran Shanmugam
Keyword(s):  
Toxoplasma Gondii ◽  
Atp Synthase ◽  
Functional Characterization ◽  
Structural Features ◽  
Subunit Composition ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Apicomplexan Parasites ◽  
Fundamental Understanding

AbstractThe mitochondrial F-type ATP synthase, a multi-subunit nanomotor, is critical for maintaining cellular ATP levels. In Toxoplasma gondii and other apicomplexan parasites, many subunit components, necessary for proper assembly and functioning of this enzyme, appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomeric (~600 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits, a, b and d, can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex could facilitate the development of novel anti-parasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.

Abstract 546: Characterization of the I4399M Variant of Apolipoprotein(a): Implications for Altered Prothrombotic Properties of Lipoprotein(a)

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jackson McAiney ◽  
Corey Scipione ◽  
Daniel Simard ◽  
James Gauld ◽  
Michael Boffa ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Fibrin Clot ◽  
Mass Spectrometry Analysis ◽  
Clot Lysis ◽  
Elevated Plasma ◽  
Plasma Clot ◽  
Lipoprotein A ◽  
Spectrometry Analysis ◽  
Apolipoprotein A

Elevated plasma levels of lipoprotein(a) (Lp(a)) are a causal risk factor for CHD. Lp(a) closely resembles LDL, but contains an additional glycoprotein apolipoprotein(a) (apo(a)) that is structurally homologous to the fibrinolytic proenzyme plasminogen. Key sequence differences render the protease-like domain in apo(a) catalytically inactive. This has led to speculation that Lp(a) can oppose the fibrinolytic functions of plasminogen. A single nucleotide polymorphism (SNP) in the LPA gene encoding apo(a) results in an Ile to Met substitution at position 4399 in the protease-like domain. In population studies, this variant has been correlated with elevated plasma Lp(a) levels and with higher CHD risk, an effect that has been suggested to arise from effects of the variant on fibrin clot properties. We undertook a functional characterization of the effect of the I4399M substitution in apo(a). Molecular dynamics simulations of wild-type (wt) apo(a) and the Met variant revealed a shift from a buried (Ile) to slightly exposed (Met) environment, allowing for potential modification of the Met. Indeed, MALDI-TOF mass spectrometry analysis demonstrated the presence of a methionine sulfoxide moiety at this position in the Met variant. When 17-kringle recombinant forms of apo(a) were included in a plasma clot lysis assay, both the wt apo(a) and Met variant inhibited lysis, but the Met variant had a 50% greater effect. However, the Met variant was equally as efficient as wt apo(a) in inhibiting plasminogen activation on a fibrin surface. Apo(a) was also able to significantly shorten coagulation time for clots made from either purified fibrin or lipoprotein-deficient plasma, with the Met variant having twice as large an effect as wt apo(a). Morever, the Met variant resulted in greater turbidity of the clots whereas wt apo(a) had no effect. In agreement with these findings, SEM and confocal microscopy of fibrin clots showed that compared to wt apo(a), the I4399M variant resulted in significant alteration of the fibrin network, with a 4-fold increase in fibrin fiber width. Together, our data suggest that the Met4399 variant differs structurally from wt apo(a), which may underlie key differences related to its effects on fibrin clot architecture and fibrinolysis.

Identification and functional characterization of a sesquiterpene synthase gene from Eleutherococcus trifoliatus

Holzforschung ◽  
2012 ◽  
Vol 66 (2) ◽  
Author(s):  
Chi-Hsiang Wen ◽  
Yen-Hsueh Tseng ◽  
Fang-Hua Chu
Keyword(s):  
Functional Characterization ◽  
Mass Spectrometry Analysis ◽  
Terpene Synthase ◽  
Gas Chromatography Mass Spectrometry ◽  
Sesquiterpene Synthase ◽  
Reading Frame ◽  
Spectrometry Analysis ◽  
Authentic Standard ◽  
The Common

Abstract In the present study, one sesquiterpene synthase gene in Eleutherococcus trifoliatus was identified and characterized. Full-length cDNA was obtained from stems. It contained an open reading frame of 1671 bp (EtCop) with a predicted molecular mass of 64.5 kDa. The amino acid sequence of EtCop contained the common terpene synthase family motifs RR(x)8W, RxR and DDxxD. The recombinant protein from Escherichia coli was incubated with farnesyl diphosphate in order to identify the function of EtCop. The product of EtCop could be identified as an α-copaene by means of gas chromatography-mass spectrometry analysis and comparison with an authentic standard.

scholarly journals Characterization of the Widely Distributed Novel ECF42 Group of Extracytoplasmic Function σ Factors inStreptomyces venezuelae

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Qiang Liu ◽  
Daniela Pinto ◽  
Thorsten Mascher
Keyword(s):  
Signal Transduction ◽  
Fundamental Principle ◽  
Genomic Context ◽  
Content Type ◽  
Two Component ◽  
Genes Encoding ◽  
Σ Factor ◽  
The One ◽  
Target Promoter

ABSTRACTExtracytoplasmic function σ factors (ECFs) represent the third most abundant fundamental principle of bacterial signal transduction, outranked only by one- and two-component systems. A recent census of ECFs revealed a large number of novel groups whose functions and regulatory mechanisms have not yet been elucidated. Here, we report the characterization of members of the novel group ECF42. ECF42 is a highly abundant and widely distributed ECF group that is present in 11 phyla but is predominantly found inActinobacteria. Analysis of the genomic context conservation did not identify a putative anti-σ factor. Instead, ECF42 genes are cotranscribed with genes encoding a conserved DGPF protein. We have experimentally verified the target promoter of these ECFs (TGTCGA in the −35 region and CGA/TC in the −10 region), which was found upstream of the ECF42-encoding operons inStreptomyces venezuelae, suggesting that ECF42s are positively autoregulated. RNA sequencing (RNA-seq) was performed to define the regulons of the three ECF42 proteins inS. venezuelae, which identified mostly genes encoding DGPF proteins. In contrast to typical ECFs, ECF42 proteins harbor a long C-terminal extension, which is crucial for their activity. Our work provides the first analysis of the function and regulatory mechanism of this novel ECF group that contains a regulatory C-terminal extension.IMPORTANCEIn contrast to the one- and two-component signal transduction systems in bacteria, the importance and diversity of ECFs have only recently been recognized in the course of comprehensive phylogenetic and comparative genomics studies. Thus, most of the ECFs still have not been experimentally characterized regarding their physiological functions and regulation mechanisms so far. The physiological roles, target promoter, and target regulons of a novel group of ECFs, ECF42, inS. venezuelaehave been investigated in this work. More importantly, members of this group are characterized by a C-terminal extension, which has been verified to harbor a regulatory role in ECF42s. Hence, our work provides an important source for further research on such C-terminal extension containing ECFs.

scholarly journals Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

2010 ◽  
Vol 9 (11) ◽  
pp. 1650-1660 ◽  
Author(s):  
Encarnación Dueñas-Santero ◽  
Ana Belén Martín-Cuadrado ◽  
Thierry Fontaine ◽  
Jean-Paul Latgé ◽  
Francisco del Rey ◽  
...  
Keyword(s):  
Cell Wall ◽  
Schizosaccharomyces Pombe ◽  
Protein Characterization ◽  
Wall Material ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Hydrolysis Of ◽  
Controlled Hydrolysis ◽  
Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Charles T. Lauhon
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Genomic Context ◽  
Similar Reduction ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Fertile Ground ◽  
And Function

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

scholarly journals Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

2019 ◽  
Vol 116 (15) ◽  
pp. 7333-7342 ◽  
Author(s):  
Xiang Ye ◽  
Jiabei Lin ◽  
Leland Mayne ◽  
James Shorter ◽  
S. Walter Englander
Keyword(s):  
Hydrogen Exchange ◽  
Atp Hydrolysis ◽  
Physiological Solution ◽  
Mass Spectrometry Analysis ◽  
Structural Element ◽  
Spectrometry Analysis ◽  
Exchange Mass Spectrometry ◽  
Substrate Protein ◽  
Central Pore ◽  
The One

Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.

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