A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway.

Autor: Lancaster EB; Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, TX, 78712, USA., Johnson WH Jr; Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, TX, 78712, USA., LeVieux JA; Department of Molecular Biosciences, and University of Texas, Austin, TX, 78712, USA., Hardtke HA; Department of Molecular Biosciences, and University of Texas, Austin, TX, 78712, USA., Zhang YJ; Department of Molecular Biosciences, and University of Texas, Austin, TX, 78712, USA; Institute for Cellular and Molecular Biology, University of Texas, Austin, TX, 78712, USA., Whitman CP; Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, TX, 78712, USA; Institute for Cellular and Molecular Biology, University of Texas, Austin, TX, 78712, USA. Electronic address: whitman@austin.utexas.edu.
Jazyk: angličtina
Zdroj: Archives of biochemistry and biophysics [Arch Biochem Biophys] 2023 Jan 01; Vol. 733, pp. 109471. Date of Electronic Publication: 2022 Nov 26.
DOI: 10.1016/j.abb.2022.109471
Abstrakt: NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO 2 - ) 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 3 , 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.
(Copyright © 2022 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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