Characterization of a novel aromatic ring-hydroxylating oxygenase, NarA2B2, from thermophilic Hydrogenibacillus sp. strain N12

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are harmful to human health due to their carcinogenic, teratogenic, and mutagenic effects. A thermophilic Hydrogenibacillus sp. strain N12 capable of degrading a variety of PAHs and derivatives was previously isolated. In this study, an aromatic ring-hydroxylating oxygenase, NarA2B2, was identified from strain N12, with substrate specificity including naphthalene, phenanthrene, dibenzothiophene, fluorene, acenaphthene, carbazole, biphenyl, and pyrene. NarA2B2 was proposed to add one or two atoms of molecular oxygen to the substrate and catalyze biphenyl at C-2, 2 or C-3, 4 positions with different characteristics than before. The key catalytic amino acids, H222, H227, and D379, were identified as playing a pivotal role in the formation of the 2-his-1-carboxylate facial triad. Furthermore, we conducted molecular docking and molecular dynamics simulations, notably, D219 enhanced the stability of the iron center by forming two stable hydrogen bonds with H222, while the mutation of F216, T223, and H302 modulated the catalytic activity by altering the pocket's size and shape. Compared to the wild-type (WT) enzyme, the degradation ratios of acenaphthene by F216A, T223A, and H302A had an improvement of 23.08%, 26.87%, and 29.52%, the degradation ratios of naphthalene by T223A and H302A had an improvement of 51.30% and 65.17%, while the degradation ratio of biphenyl by V236A had an improvement of 77.94%. The purified NarA2B2 was oxygen-sensitive when it was incubated with L-ascorbic acid in an anaerobic environment, and its catalytic activity was restored in vitro. These results contribute to a better understanding of the molecular mechanism responsible for PAHs' degradation in thermophilic microorganisms.IMPORTANCE(i) A novel aromatic ring-hydroxylating oxygenase named NarA2B2, capable of degrading multiple polycyclic aromatic hydrocarbons and derivatives, was identified from the thermophilic microorganism Hydrogenibacillus sp. N12. (ii) The degradation characteristics of NarA2B2 were characterized by adding one or two atoms of molecular oxygen to the substrate. Unlike the previous study, NarA2B2 catalyzed biphenyl at C-2, 2 or C-3, 4 positions. (iii) Catalytic sites of NarA2B2 were conserved, and key amino acids F216, D219, H222, T223, H227, V236, F243, Y300, H302, W316, F369, and D379 played pivotal roles in catalysis, as confirmed by protein structure prediction, molecular docking, molecular dynamics simulations, and point mutation.

Keywords: Hydrogenibacillus; aromatic ring-hydroxylating oxygenase; biodegradation; polycyclic aromatic hydrocarbons.

Fig 1

Neighbor-joining phylogenetic tree of amino acid sequences of NarA2 (A) and NarB2 (B) in strain N12. The phylogenetic tree of NarAa is based on a novel RHO classification system that classifies RHOs into five types derived from the ETC components (30). The phylogenetic tree of NarB2 is constructed from homologous sequences aligned by the National Center for Biotechnology Information (NCBI).

Fig 2

Detection of substrate specificity of NarA2B2 in E. coli BL21(DE3). (A) NAP (50 mg/L); (B) PHE (50 mg/L); (C) FLN (50 mg/L); (D) ACE (50 mg/L); (E) CA (50 mg/L); (F) DBT (50 mg/L); (G) PYE (10 mg/L); and (H) BP (50 mg/L). The yellow line with circles represents E. coli BL21(DE3) containing pETDuet-narA2-Ter-narB2 and pACYCDuet-phtAcAd, and the green line with squares represents E. coli BL21(DE3) containing pETDuet and pACYCDuet. The concentration in parentheses is the initial concentration. All the tests above were conducted in triplicate. NAP, naphthalene; PHE, phenanthrene; FLN, fluorene; ACE, acenaphthene; CA, carbazole; DBT, dibenzothiophene; PYE, pyrene; and BP, biphenyl.

Fig 3

GC-MS profile of degradation samples of PAHs and their derivatives by E. coli BL21(DE3) with heterologous expression of NarA2B2 and PhtAcAd. The sample at 0 h was used as the control, and the samples at 4 and 24 h were the experimental group. (A) Naphthalene (P1: 1-naphthol, TMS; P2: 2-naphthol, TMS; P3: NAP-1,2-dihydrodiol, 2TMS; P4: 1,2-dihydroxynaphthalene, 2TMS). (B1–B3) Phenanthrene (P6, P7, P9, P10, P11: monohydroxy-PHE, TMS; P5: PHE-9,10-dihydrodiol, 2TMS; P8, P12: PHE-dihydrodiol, 2TMS; P13: dihydroxyl-PHE, 2TMS). (C) Dibenzothiophene (P14: monohydroxy-DBT, TMS; P15: dibenzothiophene-S-oxide). (D) Biphenyl (P16: 4-phenylphenol, TMS; P17: 3-hydroxybiphenyl, TMS; P18: 2,2-biphenyldiol, 2TMS). (E) Carbazole (P19: monodroxy-CA, TMS). (F) Pyrene (P20: PYE-4,5-dihydrodiol, 2TMS; P21: monodroxy-PYE, TMS). (G) Acenaphthene (P22: 1-acenaphthenone; P23: 1-acenaphthenol, TMS; P24–25: 1,2-dihydroxyacenaphthene, 2TMS; P26: 1,2-dihydroxyacenaphthylene, 2TMS). (H) Fluorene (P27: 9-fluorenone; P28: 9-fluorenol, TMS; P29-31: monodroxy-FLN, TMS; P32: 2-fluorenol, TMS; P33, P35: dihydroxy-FLN, 2TMS; P34: 2-hydroxy-9-fluorenone, TMS).

Fig 4

Proposed metabolic characteristics of PAHs and derivatives catalyzed by E. coli BL21(DE3) with heterologous expression of NarA2B2 and PhtAcAd. (A) NAP; (B) PHE; (C) DBT; (D) BP; (E) CA; (F) PYE; (G) ACE; and (H) FLN. The metabolites in parentheses were inferred from the reported pathway and were not detected in this experiment.

Fig 5

Overall structure of NarA2B2. (A) NarA2B2 predicted by AlphaFold2 with aligned naphthalene 1,2-dioxygenase from Rhodococcus sp. (PDB: 2B1X); the α-subunit NarA2 is colored in light grey (and green in 2B1X) and the β-subunit NarB2 is dark gray (and cyan in 2B1X). (B) The substrate pocket of NarA2B2, with the surface of NarA2B2 colored in light purple, and the side chain of residues around the active site are shown in sticks. (C) The active site iron center of NarA2B2, distances are given in Å.

Fig 6

The degradation of PAHs and derivatives by NarA2B2 mutants in E. coli BL21(DE3) at 24 h. (A) NAP; (B) PHE; (C) DBT; (D) ACE; (E) FLN; (E) BP; (G) CA; and (H) PYE. PC: BL21(DE3) containing pETDuet-narA2-Ter-narB2 and pACYCDuet-phtAcAd without mutations; NC: BL21(DE3) containing pETDuet and pACYCDuet; M216: F216A; M219: D219A; M222: H222A; M223: T223A; M227: H227A; M236: V236A; M243: F243A; M300: Y300A; M302: H302A; M316: W316A; M369: F369A; and M379: D379A.

Fig 7

The purification and characterization of NarA2B2. (A) The purification of NarA2B2. (B) The purification of PhtAcAd. (C) The degradation of PHE by purified NarA2B2. NarA2B2: the reaction mixture contained purified NarA2B2 and PhtAcAd; CK: the reaction mixture did not contain purified NarA2B2 and PhtAcAd. (D) GC-MS profile of PHE degraded by purified NarA2B2. The sample at 0 h was used as the control, and the samples at 5 and 19 h were the experimental group (P1–P4: monohydroxy-PHE, TMS); (E) The catalytic temperature range suitable for NarA2B2 when PHE was used as the substrate; (F) The catalytic pH range suitable for NarA2B2 when PHE was used as the substrate.

Fig 8

Structural analysis of NarA2B2 complex with representative substrate. (A) Clustering structures of NarA2B2 and its mutations with NAP (top row) and ACE (bottom row). Active site residues are displayed in stick format, along with the substrate and OOH moiety, shown in stick and ball formats, respectively. Substrates (NAP and ACE) are highlighted in magenta. Attacking distances between the substrate and the OOH moiety are indicated in orange, with distances in Å. (B) Violin density plot illustrating the attacking distances during MD simulations between NarA2B2 and its mutations with either NAP (top) or ACE (bottom). The attacking distances are determined by the minimum distance between the carbon atom of the substrate and the oxygen atom bound to iron. (C) Potential π interaction distances between residues and NAP within the WT-NAP system complex. Distances are measured from the benzene ring’s center of mass within residues and NAP. The terms “Ph,” “Im,” and “Ind” denote phenyl, imidazole, and Indole rings, respectively. Data includes three parallel MD simulations, represented by red, yellow, and blue lines. (D) Active site pocket structures of NarA2B2 and its mutations, with calculated volumes (in ų) and areas (in Ų).