5ACT

W228S-Investigation of the impact from residues W228 and Y233 in the metallo-beta-lactamase GIM-1

5ACT の概要

• エントリーDOI: 10.2210/pdb5act/pdb
• 関連するPDBエントリー: 5ACP 5ACQ 5ACR 5ACS
• 分子名称: GIM-1 PROTEIN, ZINC ION (3 entities in total)
• 機能のキーワード: hydrolase, metallo-beta-lactamase, gim-1, carbapenemase, enzyme kinetics, mic
• 由来する生物種: PSEUDOMONAS AERUGINOSA
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 54936.91

構造登録者

Skagseth, S.,Carlsen, T.J.,Bjerga, G.E.K.,Spencer, J.,Samuelsen, O.,Leiros, H.-K.S. (登録日: 2015-08-17, 公開日: 2015-12-23, 最終更新日: 2024-01-10)

主引用文献

Skagseth, S.,Carlsen, T.J.,Bjerga, G.E.K.,Spencer, J.,Samuelsen, O.,Leiros, H.S.
Role of Residues W228 and Y233 in the Structure and Activity of Metallo-Beta-Lactamase Gim-1.
Antimicrob.Agents Chemother., 60:990-, 2015

PubMed Abstract: Metallo-β-lactamases (MBLs) hydrolyze virtually all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative β-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with β-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the β-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

PubMed: 26643332
DOI: 10.1128/AAC.02017-15

実験手法

X-RAY DIFFRACTION (1.81 Å)