scholarly journals Conformational communication mediates the reset step in t6A biosynthesis

2019 ◽  
Vol 47 (12) ◽  
pp. 6551-6567 ◽  
Author(s):  
Amit Luthra ◽  
Naduni Paranagama ◽  
William Swinehart ◽  
Susan Bayooz ◽  
Phuc Phan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translational Fidelity ◽  
Substrate Analog ◽  
Binding Cavity ◽  
Saxs Analysis

Abstract The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

scholarly journals Structure-Based Site-Directed Mutagenesis of the UDP-MurNAc-Pentapeptide-Binding Cavity of the FemX Alanyl Transferase from Weissella viridescens

2005 ◽  
Vol 187 (11) ◽  
pp. 3833-3838 ◽  
Author(s):  
Antoine P. Maillard ◽  
Sabrina Biarrotte-Sorin ◽  
Régis Villet ◽  
Stéphane Mesnage ◽  
Ahmed Bouhss ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bond ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Transferase Activity ◽  
Hydrogen Bond Network ◽  
Gram Positive Bacteria ◽  
Binding Cavity ◽  
Bond Network ◽  
Phosphate Groups

ABSTRACT Weissella viridescens FemX (FemXWv) belongs to the Fem family of nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor to synthesize the peptide cross-bridge found in the peptidoglycan of many species of pathogenic gram-positive bacteria. We have recently solved the crystal structure of FemXWv in complex with the peptidoglycan precursor UDP-MurNAc-pentapeptide and report here the site-directed mutagenesis of nine residues located in the binding cavity for this substrate. Two substitutions, Lys36Met and Arg211Met, depressed FemXWv transferase activity below detectable levels without affecting protein folding. Analogues of UDP-MurNAc-pentapeptide lacking the phosphate groups or the C-terminal d-alanyl residues were not substrates of the enzyme. These results indicate that Lys36 and Arg211 participate in a complex hydrogen bond network that connects the C-terminal d-Ala residues to the phosphate groups of UDP-MurNAc-pentapeptide and constrains the substrate in a conformation that is essential for transferase activity.

Structural and biochemical analyses of theStreptococcus pneumoniaeL,D-carboxypeptidase DacB

2015 ◽  
Vol 71 (2) ◽  
pp. 283-292
Author(s):  
Juan Zhang ◽  
Yi-Hu Yang ◽  
Yong-Liang Jiang ◽  
Cong-Zhao Zhou ◽  
Yuxing Chen
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Properties ◽  
Biochemical Analyses ◽  
Key Residues ◽  
Structural Insights

The L,D-carboxypeptidase DacB plays a key role in the remodelling ofStreptococcus pneumoniaepeptidoglycan during cell division. In order to decipher its substrate-binding properties and catalytic mechanism, the 1.71 Å resolution crystal structure of DacB fromS. pneumoniaeTIGR4 is reported. Structural analyses in combination with comparisons with the recently reported structures of DacB fromS. pneumoniaeD39 and R6 clearly demonstrate that DacB adopts a zinc-dependent carboxypeptidase fold and belongs to the metallopeptidase M15B subfamily. In addition, enzymatic activity assays further confirm that DacB indeed acts as an L,D-carboxypeptidase towards the tetrapeptide L-Ala-D-iGln-L-Lys-D-Ala of the peptidoglycan stem, withKmandkcatvalues of 2.84 ± 0.37 mMand 91.49 ± 0.05 s−1, respectively. Subsequent molecular docking and site-directed mutagenesis enable the assignment of the key residues that bind to the tetrapeptide. Altogether, these findings provide structural insights into substrate recognition in the metallopeptidase M15B subfamily.

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 Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

2014 ◽  
Vol 70 (4) ◽  
pp. 1166-1172 ◽  
Author(s):  
Michael E. Webb ◽  
Briony A. Yorke ◽  
Tom Kershaw ◽  
Sarah Lovelock ◽  
Carina M. C. Lobley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intramolecular Rearrangement ◽  
Directed Mutagenesis ◽  
Vitamin B ◽  
Biosynthesis Pathway ◽  
Serine Residue ◽  
Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

scholarly journals Crystal Structure and Site-directed Mutagenesis ofBacillus maceransEndo-1,31,4--glucanase

1995 ◽  
Vol 270 (7) ◽  
pp. 3081-3088 ◽  
Author(s):  
Michael Hahn ◽  
Ole Olsen ◽  
Oliver Politz ◽  
Rainer Borriss ◽  
Udo Heinemann
Keyword(s):  
Crystal Structure ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Crystal Structure and Site-directed Mutagenesis of Enzymatic Components from Clostridium perfringens Iota-toxin

2003 ◽  
Vol 325 (3) ◽  
pp. 471-483 ◽  
Author(s):  
Hideaki Tsuge ◽  
Masahiro Nagahama ◽  
Hiroyuki Nishimura ◽  
Junzo Hisatsune ◽  
Yoshihiko Sakaguchi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Clostridium Perfringens ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Clostridium Perfringens Iota Toxin

scholarly journals Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass

2020 ◽  
Vol 295 (33) ◽  
pp. 11455-11465 ◽  
Author(s):  
Julius Campeciño ◽  
Satyanarayana Lagishetty ◽  
Zdzislaw Wawrzak ◽  
Victor Sosa Alfaro ◽  
Nicolai Lehnert ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nitrite Reductase ◽  
Nitrate Reduction ◽  
Convergent Evolution ◽  
Nutrient Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cytochrome C Nitrite Reductase ◽  
Environmental Bacterium ◽  
Key Driver

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution.

Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP

2000 ◽  
Vol 349 (1) ◽  
pp. 333-342 ◽  
Author(s):  
Gyles COZIER ◽  
Richard SESSIONS ◽  
Joanna R. BOTTOMLEY ◽  
Jon S. REYNOLDS ◽  
Peter J. CULLEN
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Inositol Phosphate ◽  
Site Directed Mutagenesis ◽  
Pleckstrin Homology Domain ◽  
Directed Mutagenesis ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Gtpase Activating Protein ◽  
Ras Gtpase

GAP1IP4BP is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have studied Ins(1,3,4,5)P4 binding to GAP1IP4BP, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P4 binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P4 binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P4 has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1IP4BP. Therefore we generated a model of the PH domain of GAP1IP4BP in complex with Ins(1,3,4,5)P4 based on the Btk-Ins(1,3,4,5)P4 complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P4, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

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