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Enhancing PLP-binding capacity of class-III ω-transaminase by single residue substitution

DOI: 10.3389/fbioe.2019.00282 DOI Help

Authors: David Roura Padrosa (University of Nottingham) , Raphael Alaux (University of Nottingham) , Phillip Smith (University of Nottingham) , Ingrid Dreveny (University of Nottingham) , Fernando López-gallego (Instituto de Síntesis Química y Catálisis Homogénea; ARAID Foundation) , Francesca Paradisi (University of Nottingham; University of Bern)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Frontiers In Bioengineering And Biotechnology , VOL 7

State: Published (Approved)
Published: October 2019
Diamond Proposal Number(s): 19880

Open Access Open Access

Abstract: Transaminases are pyridoxal-5′-phosphate (PLP) binding enzymes, broadly studied for their potential industrial application. Their affinity for PLP has been related to their performance and operational stability and while significant differences in PLP requirements have been reported, the environment of the PLP-binding pocket is highly conserved. In this study, thorough analysis of the residue interaction network of three homologous transaminases Halomonas elongata (HeTA), Chromobacterium violaceum (CvTA), and Pseudomonas fluorescens (PfTA) revealed a single residue difference in their PLP binding pocket: an asparagine at position 120 in HeTA. N120 is suitably positioned to interact with an aspartic acid known to protonate the PLP pyridinium nitrogen, while the equivalent position is occupied by a valine in the other two enzymes. Three different mutants were constructed (HeTA-N120V, CvTA-V124N, and PfTA-V129N) and functionally analyzed. Notably, in HeTA and CvTA, the asparagine variants, consistently exhibited a higher thermal stability and a significant decrease in the dissociation constant (Kd) for PLP, confirming the important role of N120 in PLP binding. Moreover, the reaction intermediate pyridoxamine-5′-phosphate (PMP) was released more slowly into the bulk, indicating that the mutation also enhances their PMP binding capacity. The crystal structure of PfTA, elucidated in this work, revealed a tetrameric arrangement with the PLP binding sites near the subunit interface. In this case, the V129N mutation had a negligible effect on PLP-binding, but it reduced its temperature stability possibly destabilizing the quaternary structure.

Journal Keywords: pyridoxal phosphate; protein stability; protein engineering; biocatalysis; transaminase

Subject Areas: Biology and Bio-materials

Instruments: I24-Microfocus Macromolecular Crystallography


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