An enzymic l-2-hydroxyglutarate biosensor based on l-2-hydroxyglutarate dehydrogenase from Azoarcus olearius

Area of Science:

• Biochemistry
• Metabolomics
• Biosensor Technology

Background:

• l-2-Hydroxyglutarate (l-2-HG) is a crucial metabolite involved in signaling and immune processes.
• Elevated l-2-HG levels are associated with serious health conditions, including l-2-hydroxyglutaric aciduria and renal cancer.
• There is a significant need for sensitive and high-throughput methods for detecting l-2-HG.

Purpose of the Study:

• To identify and characterize an enzyme with high specificity for l-2-HG dehydrogenation.
• To develop a novel enzymatic assay-based fluorescent reporter for l-2-HG detection.
• To optimize the performance of the developed biosensor for sensitive and accurate l-2-HG quantitation.

Main Methods:

• Screening of ten l-2-HG dehydrogenase homologs to identify Azoarcus olearius BH72 l-2-HG dehydrogenase (AoL2HGDH) based on specificity and activity.
• Construction of an l-2-HG-sensing fluorescent reporter (EaLHGFR) by combining AoL2HGDH with resazurin.
• Systematic optimization of the EaLHGFR response magnitude and limit of detection using a single-factor screening strategy.

Main Results:

• AoL2HGDH was identified as a highly specific and active enzyme for l-2-HG dehydrogenation.
• The optimized biosensor, EaLHGFR-2, demonstrated a significant response magnitude of 2189.25 ± 26.89%.
• EaLHGFR-2 achieved a low limit of detection of 0.042 μM and accurately quantified l-2-HG in diverse biological samples, including human body fluids.

Conclusions:

• The developed EaLHGFR-2 biosensor provides a sensitive and specific method for l-2-HG detection.
• EaLHGFR-2 shows potential for accurate l-2-HG quantitation in various biological and clinical settings.
• This biosensor represents a promising alternative for the analysis of l-2-HG in biological samples.