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 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 Functional and Structural Investigation of Chalcone Synthases Based on Integrated Metabolomics and Transcriptome Analysis on Flavonoids and Anthocyanins Biosynthesis of the Fern Cyclosorus parasiticus

2021 ◽  
Vol 12 ◽  
Author(s):  
Meng Niu ◽  
Jie Fu ◽  
Rong Ni ◽  
Rui-Lin Xiong ◽  
Ting-Ting Zhu ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Structural Investigation ◽  
Catalytic Mechanism ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Qrt Pcr ◽  
Genes Encoding ◽  
Similar Conformation ◽  
Integrated Metabolomics

The biosynthesis of flavonoids and anthocyanidins has been exclusively investigated in angiosperms but largely unknown in ferns. This study integrated metabolomics and transcriptome to analyze the fronds from different development stages (S1 without spores and S2 with brown spores) of Cyclosorus parasiticus. About 221 flavonoid and anthocyanin metabolites were identified between S1 and S2. Transcriptome analysis revealed several genes encoding the key enzymes involved in the biosynthesis of flavonoids, and anthocyanins were upregulated in S2, which were validated by qRT-PCR. Functional characterization of two chalcone synthases (CpCHS1 and CpCHS2) indicated that CpCHS1 can catalyze the formation of pinocembrin, naringenin, and eriodictyol, respectively; however, CpCHS2 was inactive. The crystallization investigation of CpCHS1 indicated that it has a highly similar conformation and shares a similar general catalytic mechanism to other plants CHSs. And by site-directed mutagenesis, we found seven residues, especially Leu199 and Thr203 that are critical to the catalytic activity for CpCHS1.

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.

Structures of AzrA and of AzrC complexed with substrate or inhibitor: insight into substrate specificity and catalytic mechanism

2014 ◽  
Vol 70 (2) ◽  
pp. 553-564 ◽  
Author(s):  
Jian Yu ◽  
Daiki Ogata ◽  
ZuoQi Gai ◽  
Seiichi Taguchi ◽  
Isao Tanaka ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Crystal Structures ◽  
Azo Dyes ◽  
Catalytic Mechanism ◽  
Initial Step ◽  
Site Directed Mutagenesis ◽  
Flavin Mononucleotide ◽  
Catalytic Mechanisms ◽  
Hydroxy Groups ◽  
Insight Into

Azo dyes are major synthetic dyestuffs with one or more azo bonds and are widely used for various industrial purposes. The biodegradation of residual azo dyesviaazoreductase-catalyzed cleavage is very efficient as the initial step of wastewater treatment. The structures of the complexes of azoreductases with various substrates are therefore indispensable to understand their substrate specificity and catalytic mechanism. In this study, the crystal structures of AzrA and of AzrC complexed with Cibacron Blue (CB) and the azo dyes Acid Red 88 (AR88) and Orange I (OI) were determined. As an inhibitor/analogue of NAD(P)H, CB was located on top of flavin mononucleotide (FMN), suggesting a similar binding manner as NAD(P)H for direct hydride transfer to FMN. The structures of the AzrC–AR88 and AzrC–OI complexes showed two manners of binding for substrates possessing a hydroxy group at theorthoor theparaposition of the azo bond, respectively, while AR88 and OI were estimated to have a similar binding affinity to AzrC from ITC experiments. Although the two substrates were bound in different orientations, the hydroxy groups were located in similar positions, resulting in an arrangement of electrophilic C atoms binding with a proton/electron-donor distance of ∼3.5 Å to N5 of FMN. Catalytic mechanisms for different substrates are proposed based on the crystal structures and on site-directed mutagenesis analysis.

scholarly journals Functional characterization of alpha-glucan,water dikinase, the starch phosphorylating enzyme

2004 ◽  
Vol 377 (2) ◽  
pp. 525-532 ◽  
Author(s):  
René MIKKELSEN ◽  
Lone BAUNSGAARD ◽  
Andreas BLENNOW
Keyword(s):  
Reaction Mechanism ◽  
Functional Characterization ◽  
Fold Increase ◽  
Degree Of Polymerization ◽  
Site Directed Mutagenesis ◽  
Histidine Residue ◽  
Branch Points ◽  
Sequence Alignments ◽  
Pyruvate Phosphate Dikinase ◽  
Strict Requirement

GWD (α-glucan,water dikinase) is the enzyme that catalyses the phosphorylation of starch by a dikinase-type reaction in which the β-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glycosyl residue of amylopectin. GWD shows similarity in both sequence and reaction mechanism to bacterial PPS (pyruvate,water dikinase) and PPDK (pyruvate,phosphate dikinase). Amino acid sequence alignments identified a conserved histidine residue located in the putative phosphohistidine domain of potato GWD. Site-directed mutagenesis of this histidine residue resulted in an inactive enzyme and loss of autophosphorylation. Native GWD is a homodimer and shows a strict requirement for the presence of α-1,6 branch points in its polyglucan substrate, and exhibits a sharp 20-fold increase in activity when the degree of polymerization is increased from 27.8 to 29.5. In spite of the high variability in the degree of starch phosphorylation, GWD proteins are ubiquitous in plants. The overall reaction mechanism of GWD is similar to that of PPS and PPDK, but the GWD family appears to have arisen after divergence of the plant kingdom. The nucleotide-binding domain of GWD exhibits a closer phylogenetic relationship to prokaryotic PPSs than to PPDKs.

scholarly journals Biochemical and structural characterization of TraE from the plasmid pKM101

2014 ◽  
Vol 70 (a1) ◽  
pp. C576-C576
Author(s):  
Bastien Casu ◽  
Jonathan Smart ◽  
Mark Smith ◽  
Jurgen Sygusch ◽  
Christian Baron
Keyword(s):  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Protein Structures ◽  
Site Directed Mutagenesis ◽  
Crystallographic Structure ◽  
Dna Uptake ◽  
Secretion Systems ◽  
Wide Range ◽  
Brucella Suis ◽  
Plasmid Pkm101

In all organisms, secretion systems mediate the passage of macromolecules across cellular membranes. The bacterial type IV secretion system (T4SS) family can be divided into three functional groups. First, as typified by the Brucella suis system, T4SSs deliver effector macromolecules into eukaryotic cells during the course of infection. Second, in some Gram-negative bacteria, such as in Helicobacter pylori (ComB system), T4SSs mediate DNA uptake from and release into the extracellular environment. Thirdly, as in the IncN plasmid pKM101, T4SSs can mediate the conjugative transfer of plasmid DNA or transposons into a wide range of bacterial species. This conjugation phenomenon contributes to the spread of antibiotic resistance genes among pathogenic bacteria, leading to the emergence of multidrug-resistant pathogenic strains. TraE of the IncN plasmid pKM101 belongs to the VirB8 family of proteins, an essential component of most T4SSs that form functional dimmers in the T4SS core. Here, we present the X-ray crystallographic structure of the periplasmic domain of TraE at 2.4 Å resolution. The structure shows many similarities to the known VirB8-like protein structures from Brucella suis [1] and Agrobacterium tumefaciens [2]. However, the nature and the number of residues implicated in the dimerization interface differ considerably from those in the TraE structure [2]. Similar to other VirB8 homologs we have shown by analytical gel filtration that there is a concentration dependant equilibrium between monomeric and dimeric forms of TraE. Moreover, using a bacterial two-hybrid assay, in vivo dimerization has been demonstrated with full-length TraE and key residues for dimerization were identified by site-directed mutagenesis. Our work adds novel insights into the growing body of knowledge on VirB8-like proteins and it will inform future strategies aimed at developing inhibitors of TraE protein interactions and of plasmid transfer.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jothi K. Yuvaraj ◽  
Rebecca E. Roberts ◽  
Yonathan Sonntag ◽  
Xiao-Qing Hou ◽  
Ewald Grosse-Wilde ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Bark Beetle ◽  
Pest Control ◽  
Binding Sites ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Odorant Receptors ◽  
Functional Evolution ◽  
General Importance ◽  
Insight Into

Abstract Background Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

scholarly journals Streptococcus suis serotyping by a new multiplex PCR

2014 ◽  
Vol 63 (6) ◽  
pp. 824-830 ◽  
Author(s):  
Anusak Kerdsin ◽  
Yukihiro Akeda ◽  
Rujirat Hatrongjit ◽  
Unchaya Detchawna ◽  
Tsutomu Sekizaki ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Bacterial Species ◽  
Streptococcus Suis ◽  
Cross Reaction ◽  
The Third ◽  
Dna Mixture

A multiplex PCR was developed to detect all true serotypes of Streptococcus suis. This multiplex PCR was composed of four reaction sets. The first set identified nine serotypes (serotypes 1/2, 1, 2, 3, 7, 9, 11, 14 and 16), the second set identified eight serotypes (serotypes 4, 5, 8, 12, 18, 19, 24 and 25), the third set identified seven serotypes (serotypes 6, 10, 13, 15, 17, 23 and 31), and the last set identified five serotypes (serotypes 21, 27, 28, 29 and 30). This assay correctly detected serotypes 2, 5, 14 and 24 in human isolates, and serotypes 1, 2, 1/2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 16, 17, 19, 24, 28 and 31 in pig isolates from Thailand. No cross-reaction was observed with other bacterial species. Our multiplex PCR was able to simultaneously amplify a DNA mixture of reference Streptococcus suis serotypes. This assay should be useful for serotype surveillance of human and pig isolates of Streptococcus suis.

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