# Structural and functional analysis of Bacillus sarcosine oxidase and its activity toward cyclic imino acids

## TL;DR

This study explores how a bacterial enzyme reacts with specific amino acids and how modifying it can improve its activity.

## Contribution

The study reveals how enzyme mutations enhance reactivity toward cyclic imino acids by altering the active site structure.

## Key findings

- Tyr254 mutations increase enzyme activity by removing spatial interference at the active site.
- Altering Tyr254 disrupts charge transfer and substrate inhibition with l-thioproline.
- A water network near Tyr254 influences enzyme-substrate interactions.

## Abstract

This study investigated the reactivity of sarcosine oxidase (Sox) toward minor substrates through kinetic and structural analyses, along with mutational engineering to elucidate their reaction mechanisms. Sarcosine oxidase from Bacillus sp. (SoxB) recognizes the cyclic imino acids l‐proline (l‐Pro), d‐proline (d‐Pro), and l‐thioproline (l‐Tpr) as minor substrates. The reaction behavior varied depending on the substrates; notably, the absorption spectrum of l‐Tpr exhibited charge transfer, which was characteristic of substrate inhibition. Crystal structures of the enzyme–substrate complexes suggested that Tyr254 causes spatial interference with cyclic imino acids at the active site. The Tyr254Ala and Tyr254Gly mutants exhibited enhanced reactivity toward cyclic imino acids by eliminating this spatial interference. Crystallographic analysis of the mutants revealed an enlarged active site, which facilitated reactions with five‐membered cyclic imino acids. These mutations disrupted the electron delocalization associated with l‐Tpr, thereby eliminating charge transfer and substrate inhibition. A water network was also identified near the enzyme's active site, interacting with the side chain of Tyr254. These findings provide valuable insights into substrate specificity and may facilitate the development of enzymes with broader substrate scope and enhanced catalytic activity.

Sarcosine oxidase from Bacillus sp. (SoxB) recognizes l‐proline (l‐Pro), d‐proline (d‐Pro), and l‐thioproline (l‐Tpr) as minor substrates. In this study, we measured their absorption spectra, determined the crystal structures of their enzyme complexes, and performed site‐directed mutagenesis of key residues. These approaches successfully enhanced the enzyme's activity toward the cyclic imino acids. The findings provide valuable insights for improving enzyme performance.