scholarly journals Mycobacterial OtsA structures unveil substrate preference mechanism and allosteric regulation by 2-oxoglutarate and 2-phosphoglycerate

2019 ◽  
Author(s):  
Vítor Mendes ◽  
Marta Acebrón-García-de-Eulate ◽  
Nupur Verma ◽  
Michal Blaszczyk ◽  
Márcio V. B. Dias ◽  
...  
Keyword(s):  
Cell Wall ◽  
Feedback Inhibition ◽  
Structural Information ◽  
Site Directed Mutagenesis ◽  
Substrate Preference ◽  
Directed Mutagenesis ◽  
Allosteric Site ◽  
Trehalose Biosynthesis ◽  
Key Residues ◽  
Allosteric Regulators

AbstractTrehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential inM. tuberculosis, with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Herein, we report the first mycobacterial OtsA structures fromM. thermoresistibilein both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme and map a new allosteric site.

scholarly journals Mycobacterial OtsA Structures Unveil Substrate Preference Mechanism and Allosteric Regulation by 2-Oxoglutarate and 2-Phosphoglycerate

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Vítor Mendes ◽  
Marta Acebrón-García-de-Eulate ◽  
Nupur Verma ◽  
Michal Blaszczyk ◽  
Márcio V. B. Dias ◽  
...  
Keyword(s):  
Feedback Inhibition ◽  
Structural Information ◽  
Cell Envelope ◽  
Site Directed Mutagenesis ◽  
Substrate Preference ◽  
Allosteric Site ◽  
Content Type ◽  
Mycolic Acids ◽  
Trehalose Biosynthesis ◽  
Key Residues

ABSTRACT Trehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential in Mycobacterium tuberculosis, with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Here, we report the first mycobacterial OtsA structures from Mycobacterium thermoresistibile in both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme, and map a new allosteric site. IMPORTANCE Mycobacterial infections are a significant source of mortality worldwide, causing millions of deaths annually. Trehalose is a multipurpose disaccharide that plays a fundamental structural role in these organisms as a component of mycolic acids, a molecular hallmark of the cell envelope of mycobacteria. Here, we describe the first mycobacterial OtsA structures. We show mechanisms of substrate preference and show that OtsA is regulated allosterically by 2-oxoglutarate and 2-phosphoglycerate at an interfacial site. These results identify a new allosteric site and provide insight on the regulation of trehalose synthesis through the OtsAB pathway in mycobacteria.

Cel48A fromThermobifida fusca: Structure and site directed mutagenesis of key residues

2013 ◽  
Vol 111 (4) ◽  
pp. 664-673 ◽  
Author(s):  
Maxim Kostylev ◽  
Markus Alahuhta ◽  
Mo Chen ◽  
Roman Brunecky ◽  
Michael E. Himmel ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Key Residues

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

2011 ◽  
Vol 25 (6) ◽  
pp. 1874-1886 ◽  
Author(s):  
Roberta Cozzi ◽  
Enrico Malito ◽  
Annalisa Nuccitelli ◽  
Mariapina D'Onofrio ◽  
Manuele Martinelli ◽  
...  
Keyword(s):  
Structure Analysis ◽  
Group B Streptococcus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Group B ◽  
Key Residues

Site-directed mutagenesis studies on the uridine monophosphate binding sites of feedback inhibition in carbamoyl phosphate synthetase and effects on cytidine production by Bacillus amyloliquefaciens

2013 ◽  
Vol 59 (6) ◽  
pp. 374-379 ◽  
Author(s):  
Haitian Fang ◽  
Huiyan Liu ◽  
Ning Chen ◽  
Chenglin Zhang ◽  
Xixian Xie ◽  
...  
Keyword(s):  
Binding Sites ◽  
Bacillus Amyloliquefaciens ◽  
De Novo ◽  
Feedback Inhibition ◽  
Large Subunit ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Uridine Monophosphate

A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP’s feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains.

Structure and allosteric regulation of eukaryotic 6-phosphofructokinases

2013 ◽  
Vol 394 (8) ◽  
pp. 977-993 ◽  
Author(s):  
Torsten Schöneberg ◽  
Marco Kloos ◽  
Antje Brüser ◽  
Jürgen Kirchberger ◽  
Norbert Sträter
Keyword(s):  
Crystal Structures ◽  
Structural Information ◽  
Current Knowledge ◽  
Allosteric Regulation ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Special Focus ◽  
Molecular Architecture ◽  
Key Enzyme

Abstract Although the crystal structures of prokaryotic 6-phosphofructokinase, a key enzyme of glycolysis, have been available for almost 25 years now, structural information about the more complex and highly regulated eukaryotic enzymes is still lacking until now. This review provides an overview of the current knowledge of eukaryotic 6-phosphofructokinase based on recent crystal structures, kinetic analyses and site-directed mutagenesis data with special focus on the molecular architecture and the structural basis of allosteric regulation.

scholarly journals Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity

2020 ◽  
Vol 295 (11) ◽  
pp. 3734-3745
Author(s):  
Chia-Yu Kang ◽  
I-Hsiu Huang ◽  
Chi-Chi Chou ◽  
Tsai-Yu Wu ◽  
Jyun-Cyuan Chang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Catalytic Activity ◽  
Clostridium Difficile ◽  
Substrate Specificity ◽  
Surface Proteins ◽  
Site Directed Mutagenesis ◽  
Serine Residue ◽  
Peptidoglycan Cell Wall ◽  
Attractive Therapeutic Target ◽  
Key Residues

Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus. The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB–substrate complexes. Furthermore, we also demonstrated that residues 163–168 located on the β6/β7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

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