Crystal Structure of α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase: Insight into the Active Site and Catalytic Mechanism of a Novel Decarboxylation Reaction†,‡

The ammonia-producing arginine succinyltransferase pathway is the major pathway inEscherichia coliand related bacteria for arginine catabolism as a sole nitrogen source. This pathway consists of five steps, each catalyzed by a distinct enzyme. Here we report the crystal structure ofN-succinylarginine dihydrolase AstB, the second enzyme of the arginine succinyltransferase pathway, providing the first structural insight into enzymes from this pathway. The enzyme exhibits a pseudo 5-fold symmetric α/β propeller fold of circularly arranged ββαβ modules enclosing the active site. The crystal structure indicates clearly that this enzyme belongs to the amidinotransferase (AT) superfamily and that the active site contains a Cys–His-Glu triad characteristic of the AT superfamily. Structures of the complexes of AstB with the reaction product and a C365S mutant with bound theN-succinylarginine substrate suggest a catalytic mechanism that consists of two cycles of hydrolysis and ammonia release, with each cycle utilizing a mechanism similar to that proposed for arginine deiminases. Like other members of the AT superfamily of enzymes, AstB possesses a flexible loop that is disordered in the absence of substrate and assumes an ordered conformation upon substrate binding, shielding the ligand from the bulk solvent, thereby controlling substrate access and product release.

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The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714023827 ◽

2014 ◽

Vol 70 (12) ◽

pp. 3212-3225 ◽

Cited By ~ 16

Author(s):

Tiila-Riikka Kiema ◽

Rajesh K. Harijan ◽

Malgorzata Strozyk ◽

Toshiyuki Fukao ◽

Stefan E. H. Alexson ◽

...

Keyword(s):

Crystal Structure ◽

Reaction Mechanism ◽

Active Site ◽

Quaternary Structure ◽

Fatty Acyl ◽

Physiological Importance ◽

Loop Domain ◽

Solution Studies ◽

Hydrolysis Of ◽

Insight Into

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

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Automated docking of phospholipids to the phospholipase D active site: insight into the catalytic mechanism

10.31274/rtd-20200803-109 ◽

2003 ◽

Author(s):

Christopher Lynn Aikens

Keyword(s):

Phospholipase D ◽

Active Site ◽

Catalytic Mechanism ◽

Automated Docking ◽

Insight Into

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

Biochemical Journal ◽

10.1042/bj20090110 ◽

2009 ◽

Vol 420 (2) ◽

pp. 221-227 ◽

Cited By ~ 56

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012936 ◽

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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Evidence for an essential serine residue in the active site of the Theta class glutathione transferases

Biochemical Journal ◽

10.1042/bj3110247 ◽

1995 ◽

Vol 311 (1) ◽

pp. 247-250 ◽

Cited By ~ 96

Author(s):

P G Board ◽

M Coggan ◽

M C J Wilce ◽

M W Parker

Keyword(s):

Crystal Structure ◽

Active Site ◽

Catalytic Mechanism ◽

Glutathione Transferases ◽

Tyrosine Residue ◽

Serine Residue ◽

Sulphur Atom ◽

N Terminus ◽

Australian Sheep ◽

Consistent Feature

A consistent feature of the Alpha-, Mu- and Pi-class glutathione transferases (GSTs) is the presence near the N-terminus of a tyrosine residue that contributes to the activation of glutathione. While this residue appears to be conserved in many Theta-class GSTs, its absence in some suggested that the Theta-class GSTs may have a significantly different structure or catalytic mechanism. The elucidation of the crystal structure of the Theta-class GST from the Australian sheep blowfly, Lucilia cuprina, has indicated that a serine residue rather than a tyrosine residue can form a hydrogen bond with the glutathionyl sulphur atom. The present studies show that mutation of Ser-9 to alanine substantially inactivates the L. cuprina GST, confirming its importance in the reaction mechanism. As this serine is conserved in all Theta-class enzymes reported so far, it seems that an active-site serine is a significant factor that distinguishes the Theta-class GSTs from members of the Alpha-, Mu- and Pi-class isoenzymes.

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A new insight into the zinc-dependent DNA-cleavage by the colicin E7 nuclease: a crystallographic and computational study

Metallomics ◽

10.1039/c4mt00195h ◽

2014 ◽

Vol 6 (11) ◽

pp. 2090-2099 ◽

Cited By ~ 4

Author(s):

Anikó Czene ◽

Eszter Tóth ◽

Eszter Németh ◽

Harm Otten ◽

Jens-Christian N. Poulsen ◽

...

Keyword(s):

Crystal Structure ◽

Dna Cleavage ◽

Catalytic Mechanism ◽

Computational Study ◽

Colicin E7 ◽

Insight Into

The crystal structure of a colicin E7 metallonuclease mutant complemented by QM/MM calculations suggests an alternative catalytic mechanism of Zn2+-containing HNH nucleases.

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