# Identification and characterization of RacX, a new broad-specificity amino acid racemase from a novel taxon within the order Flavobacteriales

## TL;DR

Scientists discovered a new enzyme, RacX, from a salt-loving bacterium that efficiently converts amino acids and could be useful in industrial processes.

## Contribution

The discovery of a novel broad-specificity amino acid racemase, RacX, from an understudied taxon in Flavobacteriales.

## Key findings

- RacX shows high catalytic efficiency for multiple amino acids, including l-Lys and d-Lys.
- The A79C mutation increases reverse reaction efficiency by 44%, altering the enzyme's catalytic bias.
- RacX is stable under high-salt and alkaline conditions, making it suitable for industrial DAA production.

## Abstract

Amino acid racemases are pivotal for d-amino acid (DAA) biosynthesis with wide-ranging biotechnological applications, yet their industrial deployment is hindered by narrow substrate specificity and instability. Here, we report the discovery of Halocola ammonii gen. nov., sp. nov. DA487T, a novel taxon within the proposed family Halocolacceae fam. nov. (order Flavobacteriales), isolated from hypersaline sediments. Genomic analysis revealed a robust DAA metabolic network, including a putative broad-specificity racemase RacX. Biochemical characterization demonstrated RacX’s exceptional catalytic efficiency (kcat/Km = 151.2 s−1 mM−1 for l-Lys, kcat/Km = 17.8 s−1 mM−1 for d-Lys) and broad substrate spectrum (15/17 tested l-amino acids). Homology modeling and mutagenesis identified Ala79 and Cys193 as putative catalytic residues, based on structural conservation with EcL-DER. Remarkably, the A79C variant enhanced the reverse reaction efficiency (d-Lys → l-Lys) by 44%, effectively shifting the enzyme’s catalytic bias and the resulting steady-state ratio of enzyme-bound species. Computational docking suggested that Asn80, Thr81, Asn121, and Thr124 may modulate substrate binding, though experimental structural validation is required. The thermostability-lability tradeoff (T1/255°C =  70 min) highlights targets for protein engineering. Our findings not only expand the phylogenetic diversity of microbial racemases but also identify a promising biocatalyst candidate for industrial DAA production.

Microbial adaptations to extreme environments serve as a valuable source of novel biocatalysts with potential for sustainable industrial applications. In this study, we characterized Halocola ammonii DA487ᵀ, a halophilic bacterium representing the novel family Halocolaceae within the order Flavobacteriales, and identified a broad-specificity amino acid racemase, RacX. RacX demonstrates exceptional catalytic efficiency (kcat/Km up to 151.2 s⁻¹ mM⁻¹ for l-Lys) across multiple amino acids and exhibits remarkable stability under neutral and alkaline conditions (pH 7.0–9.0)—properties intrinsically linked to its high-salt ecological niche. Unlike most known racemases from neutrophilic organisms, RacX originates from an understudied phylogenetic lineage and displays unique mechanistic features, including a strong innate bias toward d-amino acid (DAA) production that can be rationally reprogrammed via single-residue substitution (e.g., A79C). These functional and evolutionary insights, combined with its halotolerance and broad substrate scope, position RacX as a promising and engineerable biocatalyst for industrial processes requiring operation under high-salt or alkaline conditions, such as the synthesis of DAA precursors for antibiotics.