# Structure and substrate promiscuity of Campylobacter jejuni periplasmic nitrate reductase (Nap) and phylogenetic analysis of Nap homologs

## TL;DR

This study reveals the structure and function of a nitrate reductase from Campylobacter jejuni, showing how it handles different substrates and relates to other similar enzymes.

## Contribution

The first cryo-EM structure of CjNapA from ε-proteobacteria and insights into its substrate promiscuity and evolutionary adaptations.

## Key findings

- CjNapA has three lysine-rich insertions that may enhance nitrate and anionic substrate affinity.
- Wild-type and C176D variants of CjNapA can reduce chlorate, perchlorate, and nitrate, but with varying efficiency.
- Structural and kinetic comparisons suggest evolutionary tuning of CjNapA for specific substrate functions.

## Abstract

Periplasmic nitrate reductase NapA is a member of the DMSO reductase (DMSOR) superfamily, which catalyzes the reduction of nitrate to nitrite. Campylobacter jejuni NapA (CjNapA) is notably larger compared to other structurally characterized NapA. Herein, we present the cryo-EM structure of CjNapA, the first of its kind from any ε-proteobacteria, revealing three lysine-rich insertions that could affect the substrate channel, potentially enhancing the affinity towards nitrate and other anionic substrates. Here, we report that wild-type CjNapA and NapA-C176D variants can reduce chlorate, perchlorate, and nitrate. However, the perchlorate and chlorate reductions by the CjNapA C176D variant are considerably slower, even though the perchlorate reductase has an Asp coordination to Mo. Molecular Dynamics (MD) simulations were performed to investigate the impact of the C176D mutation on substrate affinity and protein flexibility. Structural and kinetic comparisons with perchlorate reductase support evolutionary tuning for a desired function. Finally, structural comparisons with other structurally characterized NapAs also suggest the role of proximal pterin in CjNapA in electron transfer to the Mo center.