scholarly journals Genetic and Functional Analyses of PptA, a Phospho-Form Transferase Targeting Type IV Pili in Neisseria gonorrhoeae

2007 ◽  
Vol 190 (1) ◽  
pp. 387-400 ◽  
Author(s):  
Cecilia L. Næssan ◽  
Wolfgang Egge-Jacobsen ◽  
Ryan W. Heiniger ◽  
Matthew C. Wolfgang ◽  
Finn Erik Aas ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Metal Binding ◽  
Catalytic Mechanism ◽  
Phylogenetic Analyses ◽  
Site Directed Mutagenesis ◽  
Head Group ◽  
Sequence Alignments ◽  
Type Iv ◽  
Covalent Modifications ◽  
Division Cell

ABSTRACT The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae.

scholarly journals Probing the Essential Catalytic Residues and Substrate Affinity in the Thermoactive Bacillus stearothermophilus US100 l-Arabinose Isomerase by Site-Directed Mutagenesis

2007 ◽  
Vol 189 (9) ◽  
pp. 3556-3563 ◽  
Author(s):  
Moez Rhimi ◽  
Michel Juy ◽  
Nushin Aghajari ◽  
Richard Haser ◽  
Samir Bejar
Keyword(s):  
Amino Acids ◽  
Bacillus Stearothermophilus ◽  
Catalytic Mechanism ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Isomerization Reaction ◽  
Directed Mutagenesis ◽  
Substrate Affinity ◽  
Sequence Alignments ◽  
Arabinose Isomerase

ABSTRACT The l-arabinose isomerase (l-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 l-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 l-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the l-fucose isomerase and the l-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 l-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 l-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for l-fucose and decreased the affinity for l-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.

scholarly journals Crystal Structures of Streptococcus suis Mannonate Dehydratase (ManD) and Its Complex with Substrate: Genetic and Biochemical Evidence for a Catalytic Mechanism

2009 ◽  
Vol 191 (18) ◽  
pp. 5832-5837 ◽  
Author(s):  
Qiangmin Zhang ◽  
Feng Gao ◽  
Hao Peng ◽  
Hao Cheng ◽  
Yiwei Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Crystal Structures ◽  
Catalytic Mechanism ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Streptococcus Suis ◽  
Site Directed Mutagenesis ◽  
Crystallographic Structure ◽  
Sequence Alignments ◽  
Native Form

ABSTRACT Mannonate dehydratase (ManD) is found only in certain bacterial species, where it participates in the dissimilation of glucuronate. ManD catalyzes the dehydration of d-mannonate to yield 2-keto-3-deoxygluconate (2-KDG), the carbon and energy source for growth. Selective inactivation of ManD by drug targeting is of therapeutic interest in the treatment of human Streptococcus suis infections. Here, we report the overexpression, purification, functional characterization, and crystallographic structure of ManD from S. suis. Importantly, by Fourier transform mass spectrometry, we show that 2-KDG is formed when the chemically synthesized substrate (d-mannonate) is incubated with ManD. Inductively coupled plasma-mass spectrometry revealed the presence of Mn2+ in the purified protein, and in the solution state catalytically active ManD exists as a homodimer of two 41-kDa subunits. The crystal structures of S. suis ManD in native form and in complex with its substrate and Mn2+ ion have been solved at a resolution of 2.9 Å. The core structure of S. suis ManD is a TIM barrel similar to that of other members of the xylose isomerase-like superfamily. Structural analyses and comparative amino acid sequence alignments provide evidence for the importance of His311 and Tyr325 in ManD activity. The results of site-directed mutagenesis confirmed the functional role(s) of these residues in the dehydration reaction and a plausible mechanism for the ManD-catalyzed reaction is proposed.

scholarly journals Association of Neisseria gonorrhoeae Plasmids With Distinct Lineages and The Economic Status of Their Country of Origin

2020 ◽  
Vol 222 (11) ◽  
pp. 1826-1836 ◽  
Author(s):  
Ana Cehovin ◽  
Keith A Jolley ◽  
Martin C J Maiden ◽  
Odile B Harrison ◽  
Christoph M Tang
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Economic Status ◽  
Phylogenetic Analyses ◽  
Tetracycline Resistance ◽  
Type Iv Secretion ◽  
Conjugative Plasmid ◽  
Type Iv ◽  
High Level ◽  
Cryptic Plasmids ◽  
Chromosomal Type

Abstract Plasmids are vehicles for horizontal gene transfer between bacteria, and in Neisseria gonorrhoeae plasmids can mediate high-level antimicrobial resistance (AMR). Using genomic and phylogenetic analyses, we show that plasmids are widespread in a collection of 3724 gonococcal isolates from 56 countries, and characterized the conjugative, β-lactamase and cryptic plasmids. We found that variants of the conjugative plasmid (which can mediate tetracycline resistance) and the β-lactamase plasmid expressing TEM-135 are associated with distinct gonococcal lineages. Furthermore, AMR plasmids are significantly more prevalent in gonococci from less wealthy countries, highlighting the need for further studies. More than 94% of gonococci possess the cryptic plasmid, with its absence correlated with the presence of a novel chromosomal type IV secretion system. Our results reveal the extent of plasmid-mediated AMR in the gonococcus, particularly in less wealthy countries, where diagnostic and therapeutic options can be limited, and highlight the risk of their global spread.

Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase

2017 ◽  
Vol 474 (13) ◽  
pp. 2191-2202 ◽  
Author(s):  
Xin Fang ◽  
Jian-Xu Li ◽  
Jin-Quan Huang ◽  
You-Li Xiao ◽  
Peng Zhang ◽  
...  
Keyword(s):  
Metal Binding ◽  
Rational Design ◽  
Artemisia Annua ◽  
Complex Structure ◽  
Ring Closure ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Sequence Alignments ◽  
Terpene Synthases ◽  
And Function

Terpene synthases (TPSs) are responsible for the extremely diversified and complex structure of terpenoids. Amorpha-4,11-diene synthase (ADS) has a high (90%) fidelity in generating the sesquiterpene precursor for the biosynthesis of artemisinin, an antimalarial drug, however, little is known about how active site residues of ADS are involved in carbocation rearrangement and cyclization reactions. Here, we identify seven residues that are key to most of the catalytic steps in ADS. By structural modeling and amino acid sequence alignments of ADS with two functionally relevant sesquiterpene synthases from Artemisia annua, we performed site-directed mutagenesis and found that a single substitution, T296V, impaired the ring closure activity almost completely, and tetra-substitutions (L374Y/L404V/L405I/G439S) led to an enzyme generating 80% monocyclic bisabolyl-type sesquiterpenes, whereas a double mutant (T399L/T447G) showed compromised activity in regioselective deprotonation to yield 34.7 and 37.7% normal and aberrant deprotonation products, respectively. Notably, Thr296, Leu374, Gly439, Thr399, and Thr447, which play a major role in directing catalytic cascades, are located around conserved metal-binding motifs and function through impacting the folding of the substrate/intermediate, implying that residues surrounding the two motifs could be valuable targets for engineering TPS activity. Using this knowledge, we substantially increased amorpha-4,11-diene production in a near-additive manner by engineering Thr399 and Thr447 for product release. Our results provide new insight for the rational design of enzyme activity using synthetic biology.

scholarly journals Molecular Insights into Functional Differences between mcr-3- and mcr-1-Mediated Colistin Resistance

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Hui Li ◽  
Lu Yang ◽  
Zhihai Liu ◽  
Wenjuan Yin ◽  
Dejun Liu ◽  
...  
Keyword(s):  
Lipid A ◽  
Catalytic Mechanism ◽  
Sequence Divergence ◽  
Multidrug Resistant ◽  
Site Directed Mutagenesis ◽  
Head Group ◽  
Colistin Resistance ◽  
Flight Mass Spectrometry ◽  
Functional Differences

ABSTRACT The global emergence of plasmid-mediated colistin resistance genes mcr-1 and mcr-3 has threatened the role of the “last-resort” drug colistin in the defense against infections caused by multidrug-resistant Gram-negative bacteria. However, functional differences between these two genes in mediating colistin resistance remain poorly understood. Protein sequence alignment of MCR-3 and MCR-1 was therefore conducted in Clustal Omega to identify sequence divergence. The molecular recognition of lipid A head group phosphatidylethanolamine and MCR-3 enzyme was studied by homology modeling and molecular docking, with the catalytic mechanism of MCR-3 also being explored. Thr277 in MCR-3 was validated as the key amino acid residue responsible for the catalytic reaction using site-directed mutagenesis and was shown to act as a nucleophile. Lipid A modification induced by the MCR-3 and MCR-1 enzymes was confirmed by electrospray ionization–time of flight mass spectrometry. Far-UV circular dichroism spectra of the MCR-3 and MCR-1 enzymes suggested that MCR-3 was more thermostable than MCR-1, with a melting temperature of 66.19°C compared with 61.14°C for MCR-1. These data provided molecular insight into the functional differences between mcr-3 and mcr-1 in conferring colistin resistance.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

scholarly journals Respiratory syncytial virus B sequence analysis reveals a novel early genotype

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Juan C. Muñoz-Escalante ◽  
Andreu Comas-García ◽  
Sofía Bernal-Silva ◽  
Daniel E. Noyola
Keyword(s):  
Molecular Markers ◽  
Respiratory Syncytial Virus ◽  
Maximum Likelihood ◽  
Respiratory Infections ◽  
Phylogenetic Analyses ◽  
Detection Methods ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Individual Gene ◽  
Syncytial Virus

AbstractRespiratory syncytial virus (RSV) is a major cause of respiratory infections and is classified in two main groups, RSV-A and RSV-B, with multiple genotypes within each of them. For RSV-B, more than 30 genotypes have been described, without consensus on their definition. The lack of genotype assignation criteria has a direct impact on viral evolution understanding, development of viral detection methods as well as vaccines design. Here we analyzed the totality of complete RSV-B G gene ectodomain sequences published in GenBank until September 2018 (n = 2190) including 478 complete genome sequences using maximum likelihood and Bayesian phylogenetic analyses, as well as intergenotypic and intragenotypic distance matrices, in order to generate a systematic genotype assignation. Individual RSV-B genes were also assessed using maximum likelihood phylogenetic analyses and multiple sequence alignments were used to identify molecular markers associated to specific genotypes. Analyses of the complete G gene ectodomain region, sequences clustering patterns, and the presence of molecular markers of each individual gene indicate that the 37 previously described genotypes can be classified into fifteen distinct genotypes: BA, BA-C, BA-CC, CB1-THB, GB1-GB4, GB6, JAB1-NZB2, SAB1, SAB2, SAB4, URU2 and a novel early circulating genotype characterized in the present study and designated GB0.

β-Amyrin biosynthesis: catalytic mechanism and substrate recognition

2017 ◽  
Vol 15 (14) ◽  
pp. 2869-2891 ◽  
Author(s):  
Tsutomu Hoshino
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
The Past ◽  
Substrate Analog ◽  
Analog Experiments

In the past five years, there have been remarkable advances in the study of β-amyrin synthase. This review outlines the catalytic mechanism and substrate recognition in β-amyrin biosynthesis, which have been attained by the site-directed mutagenesis and substrate analog experiments.

scholarly journals Type IV Pilus Assembly Proficiency and Dynamics Influence Pilin Subunit Phospho-Form Macro- and Microheterogeneity in Neisseria gonorrhoeae

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e96419 ◽  
Author(s):  
Åshild Vik ◽  
Jan Haug Anonsen ◽  
Finn Erik Aas ◽  
Finn Terje Hegge ◽  
Norbert Roos ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Pilin Subunit ◽  
Pilus Assembly

Morphological redescription and molecular characterization of Trichodina matsu Basson & Van As, 1994 (Ciliophora, Mobilida, Trichodinidae) infecting Tachysurus fulvidraco (Richardson, 1846) from Chongqing, China

Zootaxa ◽  
2021 ◽  
Vol 4995 (2) ◽  
pp. 334-344
Author(s):  
QIAN ZHOU ◽  
FAHUI TANG ◽  
YUANJUN ZHAO
Keyword(s):  
Phylogenetic Analyses ◽  
Molecular Data ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Similar Species ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Multiple Sequence Alignments ◽  
Morphological And Molecular Data ◽  
Tachysurus Fulvidraco

During a survey of parasitic ciliates in Chongqing, China, Trichodina matsu Basson & Van As, 1994 was isolated from gills of Tachysurus fulvidraco. Furthermore, the 18S rRNA gene and ITS-5.8S rRNA region of T. matsu were sequenced for the first time and applied for the species identification and comparison with similar species in the present study. Based on the morphological and molecular comparisons, the results indicate that T. matsu is an ectoparasite specific for the Siluriformes catfish. Based on the analyses of genetic distance, multiple sequence alignments, and phylogenetic analyses, no obvious differentiation within populations of T. matsu was found. In addition, the ‘Trichodina hyperparasitis’ (KX904933) in GenBank is a misidentification and appears to be conspecific with T. matsu according to the comparison of morphological and molecular data.  

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