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

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

doi:10.1016/j.abb.2022.109471

Cited by 2 publications

(3 citation statements)

References 31 publications

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“…The fractions containing near-homogeneous target protein were pooled and stored at −80 °C. A sample of the purified protein was prepared as described elsewhere and analyzed by electrospray ionization mass spectrometry (ESI-MS). , …”

Section: Methodsmentioning

confidence: 99%

“…A sample of the purified protein was prepared as described elsewhere and analyzed by electrospray ionization mass spectrometry (ESI-MS). 17,18 Steady-State Kinetics of the Heterohexamer-and Fused 4-OTs. The activities of hhYR-4OT, fYR-4OT, hhCF-4OT, and fCF-4OT were examined with 2-hydroxymuconate (2-HM), 5-(methyl)-2-hydroxymuconate (5-Me-2-HM), phenylenolpyruvate (PP), 2-hydroxy-2,4-pentadienoate (HPD), and 5-(carboxymethyl)-2-hydroxymuconate (CHM) by assays described elsewhere.…”

Section: ■ Introductionmentioning

confidence: 99%

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Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases

et al. 2023

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Members of the 4-oxalocrotonate tautomerase (4-OT) subgroup in the tautomerase superfamily (TSF) are constructed from a single β–α–β unit and form homo- or heterohexamers, whereas those of the other four subgroups are composed of two consecutively joined β–α–β units and form trimers. A subset of sequences, double the length of the short 4-OTs, is found in the 4-OT subgroup. These “fused” 4-OTs form a separate subgroup that connects to the short 4-OTs in a sequence similarity network (SSN). The fused gene can be a template for the other four subgroups, resulting in the diversification of activity. Analysis of the SSN shows that multiple nodes in the fused 4-OTs connect to five linker nodes, which in turn connect to the short 4-OTs. Some fused 4-OTs are symmetric trimers and others are asymmetric trimers. The origin of this asymmetry was investigated by subjecting the sequences in three linker nodes and a closely associated fourth node to kinetic, mutagenic, and structural analyses. The results show that each sequence corresponds to the α- or β-subunit of a heterohexamer that functions as a 4-OT. Mutagenesis indicates that the key residues in both are αPro1 and βArg-11, like that of a typical 4-OT. Crystallographic analysis shows that both heterohexamers are asymmetric, where one heterodimer is flipped 180° relative to the other two heterodimers. The fusion of two subunits (α and β) of one asymmetric heterohexamer generates an asymmetric trimer with 4-OT activity. Hence, asymmetry can be introduced at the heterohexamer level and then retained in the fused trimers.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases

et al. 2023

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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

Cited by 2 publications

References 31 publications

Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases

Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases

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

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