Crystal structure of chikungunya virus nsP2 cysteine protease reveals a putative flexible loop blocking its active site

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560–564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29–131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122–131 in NAT1 model and residues 122–133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

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Crystal structure of human procathepsin X: a cysteine protease with the proregion covalently linked to the active site cysteine

Journal of Molecular Biology ◽

10.1006/jmbi.1999.3410 ◽

2000 ◽

Vol 295 (4) ◽

pp. 939-951 ◽

Cited By ~ 56

Author(s):

J Sivaraman ◽

Dorit K Nägler ◽

Rulin Zhang ◽

Robert Ménard ◽

Miroslaw Cygler

Keyword(s):

Crystal Structure ◽

Cysteine Protease ◽

Active Site ◽

Active Site Cysteine ◽

Covalently Linked

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Crystal Structure ofN-Succinylarginine Dihydrolase AstB, Bound to Substrate and Product, an Enzyme from the Arginine Catabolic Pathway ofEscherichia coli

Journal of Biological Chemistry ◽

10.1074/jbc.m413833200 ◽

2005 ◽

Vol 280 (16) ◽

pp. 15800-15808 ◽

Cited By ~ 15

Author(s):

Ante Tocilj ◽

Joseph D. Schrag ◽

Yunge Li ◽

Barbara L. Schneider ◽

Larry Reitzer ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Catalytic Mechanism ◽

Catabolic Pathway ◽

Major Pathway ◽

Product Release ◽

Flexible Loop ◽

Ammonia Release ◽

Arginine Catabolism ◽

Insight Into

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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Structures of plasmepsin II fromPlasmodium falciparumin complex with two hydroxyethylamine-based inhibitors

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15022049 ◽

2015 ◽

Vol 71 (12) ◽

pp. 1531-1539 ◽

Cited By ~ 6

Author(s):

Rosario Recacha ◽

Janis Leitans ◽

Inara Akopjana ◽

Lilija Aprupe ◽

Peteris Trapencieris ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Complex Structure ◽

Antimalarial Agents ◽

Flexible Loop ◽

Open Conformation ◽

Active Site Cavity ◽

Plasmepsin Ii ◽

Complex Structural ◽

New Compounds

Plasmepsin II (PMII) is one of the ten plasmepsins (PMs) identified in the genome ofPlasmodium falciparum, the causative agent of the most severe and deadliest form of malaria. Owing to the emergence ofP. falciparumstrains that are resistant to current antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is a constant pressure to find new and lasting chemotherapeutic drug therapies. Previously, the crystal structure of PMII in complex with NU655, a potent antimalarial hydroxyethylamine-based inhibitor, and the design of new compounds based on it have been reported. In the current study, two of these newly designed hydroxyethylamine-based inhibitors, PG418 and PG394, were cocrystallized with PMII and their structures were solved, analyzed and compared with that of the PMII–NU655 complex. Structural analysis of the PMII–PG418 complex revealed that the flap loop can adopt a fully closed conformation, stabilized by interactions with the inhibitor, and a fully open conformation, causing an overall expansion in the active-site cavity, which in turn causes unstable binding of the inhibitor. PG418 also stabilizes the flexible loop Gln275–Met286 of another monomer in the asymmetric unit of PMII, which is disordered in the PMII–NU655 complex structure. The crystal structure of PMII in complex with the inhibitor PG418 demonstrates the conformational flexibility of the active-site cavity of the plasmepsins. The interactions of the different moieties in the P1′ position of PG418 and PG394 with Thr217 have to be taken into account in the design of new potent plasmepsin inhibitors.

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Studies on a Tyr residue critical for the binding of coenzyme and substrate in mouse 3(17)α-hydroxysteroid dehydrogenase (AKR1C21): structure of the Y224D mutant enzyme

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444909051464 ◽

2010 ◽

Vol 66 (2) ◽

pp. 198-204

Author(s):

Urmi Dhagat ◽

Satoshi Endo ◽

Hiroaki Mamiya ◽

Akira Hara ◽

Ossama El-Kabbani

Keyword(s):

Crystal Structure ◽

Active Site ◽

Unique Feature ◽

Hydroxysteroid Dehydrogenase ◽

Kinetic Constants ◽

Mutant Enzyme ◽

Side Chains ◽

Wild Type ◽

Stereospecific Reduction ◽

Flexible Loop

Mouse 3(17)α-hydroxysteroid dehydrogenase (AKR1C21) is the only aldo–keto reductase that catalyzes the stereospecific reduction of 3- and 17-ketosteroids to the corresponding 3(17)α-hydroxysteroids. The Y224D mutation of AKR1C21 reduced theKmvalue for NADP(H) by up to 80-fold and completely reversed the 17α stereospecificity of the enzyme. The crystal structure of the Y224D mutant at 2.3 Å resolution revealed that the mutation resulted in a change in the conformation of the flexible loop B, including the V-shaped groove, which is a unique feature of the active-site architecture of wild-type AKR1C21 and is formed by the side chains of Tyr224 and Trp227. Furthermore, mutations (Y224F and Q222N) of residues involved in forming the safety belt for binding of the coenzyme showed similar alterations in kinetic constants for 3α-hydroxy/3-ketosteroids and 17-hydroxy/ketosteroids compared with the wild type.

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