9G66

Crystal structure of WT Rhizobium etli L-asparaginase ReAV in complex with L-Asn

9G66 の概要

• エントリーDOI: 10.2210/pdb9g66/pdb
• 関連するPDBエントリー: 7os3 7os5 7os6 7ou1 7oz6 8cly 8clz 8col 8ori 8osw 8rua 8rud 8rue 8ruf 8rug
• 分子名称: L-asparaginase II protein, 1,2-ETHANEDIOL, DI(HYDROXYETHYL)ETHER, ... (7 entities in total)
• 機能のキーワード: rhizobium etli; amidohydrolases; l-asparaginases; site-directed mutagenesis, hydrolase
• 由来する生物種: Rhizobium etli
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 81300.62

構造登録者

Pokrywka, K.,Grzechowiak, M.,Sliwiak, J.,Worsztynowicz, P.,Loch, J.I.,Ruszkowski, M.,Gilski, M.,Jaskolski, M. (登録日: 2024-07-18, 公開日: 2025-01-15, 最終更新日: 2025-03-19)

主引用文献

Pokrywka, K.,Grzechowiak, M.,Sliwiak, J.,Worsztynowicz, P.,Loch, J.I.,Ruszkowski, M.,Gilski, M.,Jaskolski, M.
Controlling enzyme activity by mutagenesis and metal exchange to obtain crystal structures of stable substrate complexes of Class 3 l-asparaginase.
Febs J., 292:1159-1173, 2025

PubMed Abstract: Rhizobium etli is a nitrogen-fixing bacterium that encodes two l-asparaginases. The structure of the inducible R. etli asparaginase ReAV has been recently determined to reveal a protein with no similarity to known enzymes with l-asparaginase activity, but showing a curious resemblance to glutaminases and β-lactamases. The uniqueness of the ReAV sequence and 3D structure make the enzyme an interesting candidate as potential replacement for the immunogenic bacterial-type asparaginases that are currently in use for the treatment of acute lymphoblastic leukemia. The detailed catalytic mechanism of ReAV is still unknown; therefore, the enzyme was subjected to mutagenetic experiments to investigate its catalytic apparatus. In this work, we generated two ReAV variants of the conserved Lys138 residue (K138A and K138H) that is involved in zinc coordination in the wild-type protein and studied them kinetically and structurally. We established that the activity of wild-type ReAV and the generated variants is significantly reduced in the presence of Cd cations, which slow down the proteins while improving their apparent substrate affinity. Moreover, the inhibitory effect of Cd is enhanced by the substitutions of Lys138, which disrupt the metal coordination sphere. The proteins with impaired activity but increased affinity were cocrystallized with the L-Asn substrate. Here, we present the crystal structures of wild-type ReAV and its K138A and K138H variants, unambiguously revealing bound l-asparagine in the active site. After careful analysis of the stereochemistry of the nucleophilic attack, we assign the role of the primary nucleophile of ReAV to Ser48. Furthermore, we propose that the reaction catalyzed by ReAV proceeds according to a double-displacement mechanism.

PubMed: 39754186
DOI: 10.1111/febs.17388

実験手法

X-RAY DIFFRACTION (1.79 Å)