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

Lancaster, Emily B.; Johnson Jr., William H.; LeVieux, Jake A.; Hardtke, Haley A.; Zhang, Yan Jessie; Whitman, Christian P.

doi:10.1016/j.abb.2022.109471

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

Journal Article · Sat Nov 26 00:00:00 EST 2022 · Archives of Biochemistry and Biophysics

DOI:https://doi.org/10.1016/j.abb.2022.109471· OSTI ID:2422628

Lancaster, Emily B. ^[1]; Johnson Jr., William H. ^[1]; LeVieux, Jake A. ^[1]; Hardtke, Haley A. ^[1]; ^[1]; ^[1]

Univ. of Texas, Austin, TX (United States)

NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO₂^-) 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. Here, 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)BH₃, 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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS)

Sponsoring Organization:: National Institutes of Health (NIH); Robert A. Welch Foundation; USDOE; USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2422628

Alternate ID(s):: OSTI ID: 1902467

Journal Information:: Archives of Biochemistry and Biophysics, Journal Name: Archives of Biochemistry and Biophysics Journal Issue: C Vol. 733; ISSN 0003-9861

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Active-site mutants
Biochemistry & Molecular Biology
Biophysics
Enzyme kinetics
Hydratase-aldolase
Naphthalene catabolic pathway
Schiff base

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

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