Recently, Prof. Hongzhi TANG and Prof. Yilei ZHAO from the School of Life Sciences and Biotechnology (SLSB), SJTU published a research article on Nature Communications, titled “Structure-guided insights into heterocyclic ring-cleavage catalysis of the non-heme Fe (II) dioxygenase NicX”. The research revealed the catalysis mechanism of the non-heme Fe(II) dioxygenase NicX.

Pyridine and its derivatives are important representative heterocyclic compounds, as pyridine rings are major constituents of natural plant alkaloids, pyridoxyl derivatives, and coenzymes such as nicotin and nicotinic acid. They are more soluble in water than are  their homocyclic analogs, and can thus be more easily transported in groundwater, which brings potentially serious health consequences. The microbial degradation pathways of nicotine and nicotinic acid have been well studied, and in both of pathways the intermediate 2, 5-dihydroxy pyridine (DHP) is produced. 2,5-Hydroxy-pyridine (DHP) is transformed to N-formylmaleamic acid (NFM) by a 2,5-DHP dioxygenase, an enzyme known as NicX from Pseudomonas putida KT2440 or Hpo from Pseudomonas putida S16. A previous biochemical study showed that this enzyme is a mononuclear non-heme iron oxygenase. Interestingly, Phylogenetic analysis of NicX and Hpo shows that these two enzymes are closely related to extradiol dioxygenases, yet there is no diol in the substrate so it can’t be called an extradiol dioxygenase, the contrast of which gives us a lot of interest in these enzymes.

Figure 1. Phylogenetic analysis of NicX. Phylogenetic tree of NicX with selected dioxygenases constructed by using neighbor-joining method. GenBank accession numbers or pdb numbers are shown at the end of each name. Bar represents 1.0 amino acid substitutions per site.

In the past five years, the researchers have devoted to solving the crystal structures of NicX and Hpo and expatiating their catalytic mechanisms to explain the abnormal phenomena mentioned above. The researchers successfully obtained the crystal structure of NicX and structure of NicX complex with the substrate DHP and product NFM. Despite extensive efforts, the research was unable to obtain a structure for Hpo. Subsequently, the article focused the efforts on the study of NicX. It is fascinating to find that NicX employs a rare four-residue (His265, Ser302, His318, and Asp320) coordination for Fe (II). Ser has been reported as a ligand for proteins including dialkylglycine decarboxylase, Cu+-ATPases, and transcriptional activators; however, it has not been reported in non-heme dioxygenases, suggesting that NicX has a previously unknown fold architecture and active site environment for non-heme iron (II) dioxygenases. The substrate is not directly coordinated with the ferrous ion of the active center; in contrast, the hydroxyl group at position 2 forms hydrogen bonds with Glu¹⁷⁷ and His¹⁸⁹, and the hydroxyl group at position 5 forms hydrogen bonds with residue His¹⁰⁵.

Figure 2. A conformational change of L104 and H105. a, In NFM bound subunits, Cys⁷⁶ is close to Leu¹⁰⁴ at a distance of 4.9 Å, away from His¹⁰⁵ at a distance of 7.7 Å. b, In DHP bound subunits, Cys⁷⁶ is close to His¹⁰⁵ at a distance of 4.1 Å, away from Leu¹⁰⁴ at a distance of 7.7 Å. Yellow, red, blue and orange represent carbon, oxygen, nitrogen and sulfur atoms, respectively.

Structural superposition comparative analysis found that a conformational change in theLeu¹⁰⁴ and His¹⁰⁵ residues induce a major change in channel II; this change creates a new hydrophobic path that goes straight to the active ferrous ion. The biochemical analyses support speculation about two possible functions of the conformational change for Leu104-His105. The first is the aforementioned creation of a hydrophobic path that could greatly facility the direct delivery of a dioxygen molecule to the iron center. The second potential function of the conformational change could be to guide and stable the substrate (DHP) to the appropriate position to initiate the ring-opening reaction to produce the product N-formylmaleamic acid (NFM)

The article proposed the different catalytic pathways of NicX, wherein the dioxygen occupies the equatorial position (Pathway I) or apical position (Pathway II), and calculated energetics of these pathways. The research can't prove which catalysis mechanism is wrong, however, the specific substrate orientation in the co-crystal structure motivated us to reconsider the possibility of Pathway II. First of all, the arrangement in Pathway II would allow maintenance of the specific hydrogen bonding network for holding the DHP substrate in place; in contrast, the DHP molecule would have to undergo a “big flip” and shift downwards by ~4 Å to bind the metal 318 and contact the equatorial dioxygen in Pathway I. Importantly, this would require disturbing the specific hydrogen bond network observed in the crystal structure. Alternatively, in Pathway II the bridging peroxo can undergo a ~0.5 Å shift of the substrate and a ~20° rotation of the substrate, a scenario that would retain the intermediate in the hydrogen bond network. Second, the first 1-electron transfer of the DHP substrate seems to occur at the moiety of O-C=N based on the pKa (pKa1 = 8.56) and Fukui function (f^- = 0.148), and the dioxygen molecule could occupy the vacancy between C6---Fe and mediate the ignition of the DHP-O2-Fe triad. Third, the consequent O-O cleavage could be promoted by neighboring proton donor candidates such as imidazolium of His¹⁰⁵, neutral form of Asp³²⁰, aromatic N-H of DHP, and even guanidinium of Arg²⁹³. Moreover, a crystal-structure-based CAVER analysis indicated that the dioxygen tunnels terminate at a position opposite to Ser³⁰² rather than Asp³²⁰.

Thus, both structural analysis and preliminary computation lend support the apicalhypothesis, but additional theoretical calculations will be required for further validation on these possible mechanisms.

It should be emphasized that both the crystal structure data and computational studies highlight differences in the apparent reaction mechanism of NicX compared to HPCD and HGDO. Specifically, (i) NicX is able to catalytically crack a pyridine ring substrate; (ii) NicX has a mononuclear iron(II) metal center that is coordinated by two histidine residues, one carboxylate and a serine residue; (iii) DHP does not directly chelate ferrous ion; (iv) the reaction between superoxide and DHP proceeds by reaction at the C6 atom of DHP, not the OH-group carrying C5 atom. In light of these differences, it is not surprising that the crystal structures and computational studies indicate clear distinctions for the proposed reaction mechanism of NicX vs. the reaction mechanisms of HPCD and HGDO. The study of NicX not only supplements the understanding of the non-heme dioxygenase, but also serves as a model for understanding other enzymes involved in the cleavage of heterocyclic compounds.

About the study in Nature Communications: (https://www.nature.com/articles/s41467-021-21567-9).

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