Promiscuous activity of ornithine decarboxylase from Enterobacter hormaechei: An alternative pathway for histamine formation in fermented sausages.

Histamine, a toxic biogenic amine in fermented foods, is classically produced by histidine decarboxylase (Hdc). Enterobacter hormaechei RH3 isolated from Sichuan fermented sausages, despite being hdc-deficient (hdc-), still accumulates high levels of histamine via ornithine decarboxylase (Odc). In this study, Hdc from Enterobacter hormaechei RH15 (hdc+) and Odc from RH3 were cloned, characterized, and compared systematically. Hdc displayed strict substrate specificity toward L-histidine with stronger affinity and higher catalytic efficiency (Km = 126.8 mM, kcat/Km = 0.00697 mM s-1; optimum 30 °C, pH 7.0). Odc showed substrate promiscuity toward both L-ornithine and L-histidine (for L-histidine: Km = 195.4 mM, kcat/Km = 0.00354 mM s-1), along with improved thermostability and pH adaptability (optimum 35 °C, pH 6.0). Molecular docking and 100 ns molecular dynamics simulations revealed distinct substrate binding modes and conformational stability, with the C-terminal domain and the positively charged active pocket potentially being the main contributors to Odc's promiscuity. Validation in sausage models demonstrated that Hdc dominated histamine formation, reaching 42 mg/kg at 120 h, while Odc acted as a secondary pathway. This study identifies a non-classical histamine synthesis pathway mediated by Odc, advances the current understanding of microbial histamine metabolism, and provides new insights into controlling biogenic amine accumulation in fermented meat products.