Staphylococcus aureusSortase Transpeptidase SrtA:  Insight into the Kinetic Mechanism and Evidence for a Reverse Protonation Catalytic Mechanism†

Biochemistry ◽  
2005 ◽  
Vol 44 (33) ◽  
pp. 11188-11200 ◽  
Author(s):  
Brenda A. Frankel ◽  
Ryan G. Kruger ◽  
Dana E. Robinson ◽  
Neil L. Kelleher ◽  
Dewey G. McCafferty
Keyword(s):  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Reverse Protonation ◽  
Insight Into

scholarly journals Structural and Kinetic Analysis of Substrate Binding to the Sialyltransferase Cst-II from Campylobacter jejuni

2011 ◽  
Vol 286 (41) ◽  
pp. 35922-35932 ◽  
Author(s):  
Ho Jun Lee ◽  
Luke L. Lairson ◽  
Jamie R. Rich ◽  
Emilie Lameignere ◽  
Warren W. Wakarchuk ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Sialic Acids ◽  
Biological Processes ◽  
Human Pathogen ◽  
Cell Surfaces ◽  
Acceptor Specificity ◽  
Wide Range ◽  
Insight Into

Sialic acids play important roles in various biological processes and typically terminate the oligosaccharide chains on the cell surfaces of a wide range of organisms, including mammals and bacteria. Their attachment is catalyzed by a set of sialyltransferases with defined specificities both for their acceptor sugars and the position of attachment. However, little is known of how this specificity is encoded. The structure of the bifunctional sialyltransferase Cst-II of the human pathogen Campylobacter jejuni in complex with CMP and the terminal trisaccharide of its natural acceptor (Neu5Ac-α-2,3-Gal-β-1,3-GalNAc) has been solved at 1.95 Å resolution, and its kinetic mechanism was shown to be iso-ordered Bi Bi, consistent with its dual acceptor substrate specificity. The trisaccharide acceptor is seen to bind to the active site of Cst-II through interactions primarily mediated by Asn-51, Tyr-81, and Arg-129. Kinetic and structural analyses of mutants modified at these positions indicate that these residues are critical for acceptor binding and catalysis, thereby providing significant new insight into the kinetic and catalytic mechanism, and acceptor specificity of this pathogen-encoded bifunctional GT-42 sialyltransferase.

Copper–oxygen Dynamics in Tyrosinase

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Sachiko Yanagisawa ◽  
Minoru Kubo ◽  
Genji Kurisu ◽  
Shinobu Itoh
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Active Form ◽  
Copper Ion ◽  
Atomic Resolution ◽  
Oxygen Species ◽  
X Ray ◽  
Oxygen Dynamics ◽  
Insight Into ◽  
Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu<sub>2</sub>O<sub>2</sub>core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.<br>

scholarly journals Crystallographic insight into the catalytic mechanism of subunit A of the A-ATP synthase and the P-loop switch in evolution

2010 ◽  
Vol 1797 ◽  
pp. 32-33
Author(s):  
Anil Kumar ◽  
Malathy Sony Subramanian Manimekalai ◽  
Asha Manikkoth Balakrishna ◽  
Gerhard Grüber
Keyword(s):  
Atp Synthase ◽  
Catalytic Mechanism ◽  
Subunit A ◽  
Insight Into

scholarly journals Alkaline ceramidase catalyzes the hydrolysis of ceramides via a catalytic mechanism shared by Zn2+-dependent amidases

2018 ◽  
Author(s):  
Jae Kyo Yi ◽  
Ruijuan Xu ◽  
Lina M. Obeid ◽  
Yusuf A. Hannun ◽  
Michael V. Airola ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Trichostatin A ◽  
Membrane Lipid ◽  
Yeast Cells ◽  
Adiponectin Receptors ◽  
Wild Type Enzyme ◽  
Zinc Coordination ◽  
Hydrolysis Of ◽  
Insight Into

ABSTRACTHuman alkaline ceramidase 3 (ACER3) is one of three alkaline ceramidases (ACERs) that catalyze the conversion of ceramide to sphingosine. ACERs are the members of the CREST superfamily of integral-membrane lipid hydrolases, including the adiponectin receptors which play roles in energy metabolism. All CREST members conserve a set of three Histidine, one Aspartate, and one Serine residue. However, the structural and catalytic roles for these residues are unclear. Here, we use ACER3 as a prototype enzyme to gain insight into this unique class of enzymes. Recombinant ACER3 was expressed in yeast cells that lack endogenous ceramidase activity, and microsomes were used for biochemical characterization. Six point mutantions of the conserved CREST motif were developed that are predicted to form a Zn-dependent active site based on homology with the human adiponectin receptors, whose crystal structures were recently determined. Five mutations completely lost their activity, except for S77A, which showed a 600-fold decrease compared with the wild-type enzyme. The activity of S77C mutation was pH sensitive, with neutral pH partially recovering ACER3 activity. This suggested a role for S77 in stabilizing the oxyanion of the transition state and differs from the proposed role in Zinc coordination for the adiponectin receptors (Vasiliauskaité-Brooks et. al., Nature, 2017). Together, these data suggest ACER3 is a Zn2+-dependent amidase that uses a catalytic mechanism for ceramide hydrolysis that is similar to other soluble Zn-based amidases. Consistent with this mechanism, ACER3 was specifically inhibited by trichostatin A, an HDAC inhibitor, which is a strong chelator of Zinc.

scholarly journals New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies

2016 ◽  
Vol 291 (29) ◽  
pp. 15057-15068 ◽  
Author(s):  
Yao Liu ◽  
João P. G. L. M. Rodrigues ◽  
Alexandre M. J. J. Bonvin ◽  
Esther A. Zaal ◽  
Celia R. Berkers ◽  
...  
Keyword(s):  
Enzyme Kinetics ◽  
Catalytic Mechanism ◽  
Docking Studies ◽  
Insight Into

Insight into structure defects and catalytic mechanism for NO oxidation over Ce0.6Mn0.4Ox solid solutions catalysts: Effect of manganese precursors

Chemosphere ◽  
2020 ◽  
Vol 243 ◽  
pp. 125406 ◽  
Author(s):  
Boyuan Hao ◽  
Yonggang Sun ◽  
Qun Shen ◽  
Xin Zhang ◽  
Zhongshen Zhang
Keyword(s):  
Solid Solutions ◽  
Catalytic Mechanism ◽  
No Oxidation ◽  
Structure Defects ◽  
Insight Into

scholarly journals GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

2009 ◽  
Vol 420 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Helge C. Dorfmueller ◽  
Vladimir S. Borodkin ◽  
Marianne Schimpl ◽  
Daan M. F. van Aalten
Keyword(s):  
Active Site ◽  
Cell Biology ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Acetyl Group ◽  
Nanomolar Range ◽  
Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

scholarly journals d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

2020 ◽  
Vol 295 (23) ◽  
pp. 7894-7904
Author(s):  
Jordan L. Pederick ◽  
Andrew P. Thompson ◽  
Stephen G. Bell ◽  
John B. Bruning
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Drug Target ◽  
Catalytic Mechanism ◽  
Thermus Thermophilus ◽  
Monovalent Cation ◽  
Antibiotic Drug ◽  
Domains Of Life ◽  
Alanine Dipeptide ◽  
Insight Into

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine–d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl–d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+. Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

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