The bacterial catabolism of polycyclic aromatic hydrocarbons: Characterization of three hydratase-aldolase-catalyzed reactions

LeVieux, Jake A.; Johnson, William H.; Erwin, Kaci; Li, Wenzong; Zhang, Yan Jessie; Whitman, Christian P.

doi:10.1016/j.pisc.2016.03.025

Cited by 3 publications

(5 citation statements)

References 14 publications

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“…The loss of PhdJ activity (in the presence of NaCNBH 3 and 6) coupled with a single covalent modification is consistent with the formation of a Schiff base between substrate and enzyme. A similar result for NahE has been previously reported (39).…”

Section: Covalent Modification Of Phdj In the Presence Of Substrate And Nacnbhsupporting

confidence: 91%

“…The common chemistry in the NAL subgroup members (as well as in the Class I aldolase superfamily) is the utilization of a Schiff base intermediate that forms between the strictly conserved lysine and the α-keto acid moiety of the substrate. , Sequence and crystallographic analysis along with Na(CN)BH 3 trapping experiments, reported here and elsewhere, support the Schiff base intermediate through Lys-183 (NahE) and Lys-180 (PhdJ). The carboxylate group of substrate 6 (in the PhdJ· 6 structure) interacts with the backbone amides of Thr-62 and Phe-63 in the conserved GTFGE motif (step 1, Scheme ).…”

Section: Resultsmentioning

confidence: 82%

“…A similar result for NahE has been previously reported. 39 1 H NMR Characterization of the PhdJ-Catalyzed Reactions. The PhdJ-catalyzed reaction using 6 was monitored by 1 H NMR spectroscopy to confirm the identities of the products.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

“…The common chemistry in the NAL subgroup members (as well as in the Class I aldolase superfamily) is the utilization of a Schiff base intermediate that forms between the strictly conserved lysine and the α-keto acid moiety of the substrate. 15,19 Sequence and crystallographic analysis along with Na(CN)BH 3 trapping experiments, reported here and elsewhere, 39 interacts with the backbone amides of Thr-62 and Phe-63 in the conserved GTFGE motif (step 1, Scheme 5). (The side chain of Glu-65 points in the opposite direction and is ∼12 Å from the ligand.)…”

mentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Covalent Modification Of Phdj In the Presence Of Substrate And Nacnbhsupporting

confidence: 91%

Section: Resultsmentioning

confidence: 82%

Section: ■ Experimental Proceduresmentioning

confidence: 99%

“…The common chemistry in the NAL subgroup members (as well as in the Class I aldolase superfamily) is the utilization of a Schiff base intermediate that forms between the strictly conserved lysine and the α-keto acid moiety of the substrate. 15,19 Sequence and crystallographic analysis along with Na(CN)BH 3 trapping experiments, reported here and elsewhere, 39 interacts with the backbone amides of Thr-62 and Phe-63 in the conserved GTFGE motif (step 1, Scheme 5). (The side chain of Glu-65 points in the opposite direction and is ∼12 Å from the ligand.)…”

mentioning

confidence: 99%

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

et al. 2018

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“…Two characteristic enzyme classes involved in PAH degradation are aromatic ring-hydroxylating dioxygenases (RHDs) and PAH hydratase-aldolases ( Martin et al, 2013 ; LeVieux et al, 2016 ; Lancaster et al, 2023 ; Yesankar et al, 2023 ). RHDs are ubiquitous in PAH degraders, catalyze the first rate-limiting step in the PAH-degradation pathway, and have a very conserved reaction site.…”

Section: Introductionmentioning

confidence: 99%

Substrate-independent expression of key functional genes in Cycloclasticus pugetii strain PS-1 limits their use as markers for PAH biodegradation

et al. 2023

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Microbial degradation of petroleum hydrocarbons is a crucial process for the clean-up of oil-contaminated environments. Cycloclasticus spp. are well-known polycyclic aromatic hydrocarbon (PAH) degraders that possess PAH-degradation marker genes including rhd3α, rhd2α, and pahE. However, it remains unknown if the expression of these genes can serve as an indicator for active PAH degradation. Here, we determined transcript-to-gene (TtG) ratios with (reverse transcription) qPCR in cultures of Cycloclasticus pugetii strain PS-1 grown with naphthalene, phenanthrene, a mixture of these PAHs, or alternate substrates (i.e., no PAHs). Mean TtG ratios of 1.99 × 10−2, 1.80 × 10−3, and 3.20 × 10−3 for rhd3α, rhd2α, and pahE, respectively, were measured in the presence or absence of PAHs. The TtG values suggested that marker-gene expression is independent of PAH degradation. Measurement of TtG ratios in Arctic seawater microcosms amended with water-accommodated crude oil fractions, and incubated under in situ temperature conditions (i.e., 1.5°C), only detected Cycloclasticus spp. rhd2α genes and transcripts (mean TtG ratio of 4.15 × 10−1). The other marker genes—rhd3α and pahE—were not detected, suggesting that not all Cycloclasticus spp. carry these genes and a broader yet-to-be-identified repertoire of PAH-degradation genes exists. The results indicate that the expression of PAH marker genes may not correlate with PAH-degradation activity, and transcription data should be interpreted cautiously.

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

Lancaster¹,

Johnson²,

LeVieux³

et al. 2023

Archives of Biochemistry and Biophysics

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The bacterial catabolism of polycyclic aromatic hydrocarbons: Characterization of three hydratase-aldolase-catalyzed reactions

Cited by 3 publications

References 14 publications

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

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

Substrate-independent expression of key functional genes in Cycloclasticus pugetii strain PS-1 limits their use as markers for PAH biodegradation

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

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