2. Academic Validation
  3. Modulating Substrate Specificity of Rhizobium sp. Histamine Dehydrogenase through Protein Engineering for Food Quality Applications

Modulating Substrate Specificity of Rhizobium sp. Histamine Dehydrogenase through Protein Engineering for Food Quality Applications

  • Molecules. 2023 Apr 26;28(9):3748. doi: 10.3390/molecules28093748.
Karen Rodríguez-Núñez 1,  Alejandra Cortés-Monroy 1,  Marcela Serey 1,  Yunus Ensari 2,  Mehdi D Davari 3,  Claudia Bernal 4,  Ronny Martinez 1
Affiliations

Affiliations

  • 1 Departamento de Ingeniería en Alimentos, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena 1720010, Chile.
  • 2 Department of Bioengineering, Faculty of Engineering and Architecture, Kafkas University, Kars 36000, Turkey.
  • 3 Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany.
  • 4 Instituto de Investigación Multidisciplinaria en Ciencia y Tecnología, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena 1720010, Chile.
PMID: 37175158 DOI: 10.3390/molecules28093748
Abstract

Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, Other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future Enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this Enzyme class.

Keywords

agmatine; dehydrogenase; diaminopropane; enzyme engineering; histamine; protein design.

Figures
Products