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

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 <i>Halocola ammonii</i> gen. nov., sp. nov. DA487^T, a novel taxon within the proposed family <i>Halocolacceae</i> fam. nov. (order <i>Flavobacteriales</i>), 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 (<i>k</i>_cat<i>/K_m</i> = 151.2 s^-1 mM^-1 for l-Lys, <i>k</i>_cat<i>/K_m</i> = 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 ([Formula: see text]) 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.IMPORTANCEMicrobial adaptations to extreme environments serve as a valuable source of novel biocatalysts with potential for sustainable industrial applications. In this study, we characterized <i>Halocola ammonii</i> DA487ᵀ, a halophilic bacterium representing the novel family <i>Halocolaceae</i> within the order <i>Flavobacteriales</i>, and identified a broad-specificity amino acid racemase, RacX. RacX demonstrates exceptional catalytic efficiency (<i>k</i>_cat<i>/K_m</i> 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.