# Structural characterization of pyruvic oxime dioxygenase, a key enzyme in heterotrophic nitrification

## TL;DR

This paper reveals the structure of a key enzyme in heterotrophic nitrification, providing insights into its function and potential for inhibitor design.

## Contribution

The study provides the first structural characterization of pyruvic oxime dioxygenase (POD) and its catalytic mechanism.

## Key findings

- The crystal structure of AfPOD reveals a homotetramer with an active site coordinated by iron and histidines.
- The N-terminal region is disordered in the crystal but adopts an α-helix conformation important for catalysis.
- A putative oxygen tunnel connects the active site to the central cavity of the tetramer.

## Abstract

Nitrification by heterotrophic microorganisms is an important part of the nitrogen cycle in the environment. The enzyme responsible for the core function of heterotrophic nitrification is pyruvic oxime dioxygenase (POD). POD is a non-heme, Fe(II)-dependent enzyme that catalyzes the dioxygenation of pyruvic oxime to produce pyruvate and nitrite. To analyze the catalytic mechanism of POD, the crystal structure of POD from Alcaligenes faecalis (AfPOD) was determined at 1.76 Å resolution. The enzyme is a homotetramer, and the subunit structure is homologous to those of class II aldolases, in particular, a zinc-dependent L-fuculose-1-phosphate aldolase. The active site of the subunit is located at the bottom of a cleft formed with an adjacent subunit. The iron ion at the active site is coordinated by three histidines and three water molecules in an octahedral geometry. The putative oxygen tunnel was connected between the active site and the central cavity of the tetramer. The N-terminal region of AfPOD, which is essential for catalytic activity, is disordered in the crystal. Structure prediction with AlphaFold2 combined with mutational experiments suggested that the disordered N-terminal region adopts an α-helix conformation and participates in the formation of the active site. The catalytic mechanism of the dioxygenase reaction by POD is discussed on the basis of the molecular docking model.

Our knowledge of nitrification has increased considerably in recent decades with the discovery of new nitrifying microorganisms and the characterization of their biochemical processes. Some heterotrophic bacteria and fungi are known to show nitrification activities, but the molecular mechanisms have been poorly understood. Here, we performed a structural characterization of pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification that produces nitrite from ammonia using pyruvic oxime as an intermediate. Structural and enzymatic analyses revealed that POD is a unique dioxygenase with features such as an aldolase backbone, an N-terminal α-helix, and an oxygen tunnel. Our results provide insights not only into the molecular mechanisms but also into the design of specific inhibitors of heterotrophic nitrification.