8DO5

Crystal structure of NahE in complex with intermediate (R)-4-hydroxy-4-(2-hydroxyphenyl)-2-iminobutanoate

8DO5 の概要

• エントリーDOI: 10.2210/pdb8do5/pdb
• 分子名称: Trans-ohydrobenzylidenepyruvate hydratase aldolase, DIMETHYL SULFOXIDE, (4R)-4-hydroxy-4-(2-hydroxyphenyl)butanoic acid, ... (4 entities in total)
• 機能のキーワード: hydratase-aldolase, tim, hydratase, aldolase, lyase
• 由来する生物種: uncultured bacterium
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 73902.53

構造登録者

LeVieux, J.A.,Hardtke, H.A.,Zhang, Y.J. (登録日: 2022-07-12, 公開日: 2022-12-28, 最終更新日: 2024-11-06)

主引用文献

Lancaster, E.B.,Johnson Jr., W.H.,LeVieux, J.A.,Hardtke, H.A.,Zhang, Y.J.,Whitman, C.P.
A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway.
Arch.Biochem.Biophys., 733:109471-109471, 2023

PubMed Abstract: NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH, and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.

PubMed: 36522814
DOI: 10.1016/j.abb.2022.109471

実験手法

X-RAY DIFFRACTION (2.1 Å)