Structural Characterization of the Hydratase-Aldolases, NahE and PhdJ: Implications for the Specificity, Catalysis, and N-Acetylneuraminate Lyase Subgroup of the Aldolase Superfamily

Abstract

NahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Because of the harmful properties of PAHs and their widespread distribution and persistence in the environment, there is great interest in understanding these degradative pathways, including the mechanisms and specificities of the enzymes found in the pathways. This knowledge can be used to develop and optimize bioremediation techniques. Although hydratase-aldolases catalyze a major step in the PAH degradative pathways, their mechanisms are poorly understood. Sequence analysis identified NahE and PhdJ as members of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. Both have a conserved lysine and tyrosine (for Schiff base formation) as well as a GXXGE motif (to bind the pyruvoyl carboxylate group). Herein, we report the structures of NahE, PhdJ, and PhdJ covalently bound to substrate via a Schiff base. Structural analysis and dynamic light scattering experiments show that both enzymes are tetramers. A hydrophobic helix insert, present in the active sites of NahE and PhdJ, might differentiate them from other NAL subgroup members. The individual specificities of NahE and PhdJ are governed by Asn-281/Glu-285 and Ser-278/Asp-282, respectively. Finally, the PhdJ complex structure suggests a potential mechanism for hydration of substrate and subsequent retro-aldol fission. The combined findings fill a gap in our mechanistic understanding of these enzymes and their place in the NAL subgroup.

Biochemical and Structural Characterization of l-2-Keto-3-deoxyarabinonate Dehydratase: A Unique Catalytic Mechanism in the Class I Aldolase Protein Superfamily

“…The secondary structural elements for AbAraD are shown above the alignment, and the colors correspond to Figure B. Highlighted active sites of AbAraD complexed with 2-oxobutyrate (B), and its superimposition with KdgD from A. tumefaciens (complex with 2-oxobutyrate) (C), KdgD from Oceanobacillus iheyensis (ligand-free) (D), HypD from S. meliloti (ligand-free) (E), the hypothetical protein from R. palustris (ligand-free) (F), KdgA from S. solfataricus [complex with d -2-keto-3-deoxygalactonate (KDGal)] (G), YagE from E. coli (complex with KDGal) (H), LRA4 from S. stipitis (complex with KDGal) (I), Hog1 from humans (ligand-free) (J), and PhdJ from M. vanbaalenii [complex with trans - o -carboxybenzylidenepyruvate (CBP)] (K). The colors of active site residues shown as sticks with carbon atoms correspond to those in panel A. Double-headed arrows with bond lengths indicate the distance between the side chain carboxyl oxygen of Glu171 in AbAraD and the side chain hydroxyl of the conserved tyrosine in other DHDPS/NAL members.…”

“…Comparisons with other structures in the PDB using PDBePISA (Proteins, Interfaces, Structures and Assemblies) clearly identified AbAraD as a member of the DHDPS/NAL protein superfamily, with strong structural similarities to many structures in “aldolase class I” (CATH superfamily 3.20.20.70). Many structures of functional DHDPS/NAL family members have been reported, including DHDPS from E. coli (1DHP; rmsd from AbAraD of 1.7 Å over 287 Cα atoms; sequence identity of 19%), NAL from E. coli (1NAL; 1.9 Å over 259 Cα atoms; 17.9%), KdgA from S. solfataricus (1W3T; 2.4 Å over 278 Cα atoms; 14%), l -KDR aldolase from Scheffersomyces stipitis (2.3 Å over 290 Cα atoms; 18%), l -KDF aldolase from Veillonella ratti (2.1 Å over 282 Cα atoms; 17%), KdgD from Agrobacterium tumefaciens (4UR8; 2.4 Å over 284 Cα atoms; 19%), trans - o -carboxybenzalpyruvate hydratase-aldolase from Mycobacterium vanbaalenii (6DAQ; 2.7 Å over 284 Cα atoms; 20%), 4-hydroxy-2-oxoglutarate aldolase from humans (3S5N; 2.2 Å over 291 Cα atoms; 19%), and Δ 1 -pyrroline-4-hydroxy-2-carboxylate deaminase from Sinorhizobium meliloti (5CZJ; 2.6 Å over 286 Cα atoms; 20%) . Despite the very low level of sequence identity with any subfamilies, structural similarities were clearly observed.…”

“…(D) Deaminase (decyclizing) for Δ 1 -pyrroline-4-hydroxy-2-carboxylate by HypD (under investigation). (E) Hydration and aldol cleavage for trans - o -carboxybenzalpyruvate by PhdJ . In panel D, the cyclic imine Δ 1 -pyrroline-4-hydroxy-2-carboxylate is spontaneously hydrolyzed to 4-hydroxy-2-oxo-5-aminovalerate.…”

“…Other enzymes of interest are trans - o -hydroxybenzylidenepyruvate hydratase-aldolase (NahE) and trans - o -carboxybenzalpyruvate hydratase-aldolase (PhdJ) . They are involved in the bacterial degradative pathways for naphthalene and phenanthrene, respectively, and catalyze the hydration reaction that sets up the retro-aldol fission (Figure ).…”

“…Other enzymes of interest are trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (NahE) and trans-o-carboxybenzalpyruvate hydratase-aldolase (PhdJ). 41 They are involved in the bacterial degradative pathways for naphthalene and phenanthrene, respectively, and catalyze the hydration reaction that sets up the retro-aldol fission (Figure 2). It is noteworthy that the imine intermediate, which is initially formed when the side chain of the conserved lysine residue makes a nucleophilic attack on the carbonyl C2 atom of the substrate, was structurally homologous to intermediate 3 in the dehydration mechanism by AbAraD (Figure 6E).…”