Development of a robust high-throughput screening system for the evaluation of human homogentisate 1,2-dioxygenase missense variants in the context of rare disease alkaptonuria

Background
Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defect homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis. HGD’s quaternary structure is known to be easily disrupted by missense mutations and this makes them an interesting target for novel treatment strategies that aim for enzyme activity rescue. However, only prediction models are available providing information on a structural basis, but not on its enzyme activities. Therefore, a whole-cell screening system in E. coli was developed to evaluate HGD missense variants.
Methods
The principle of our screening method is based on HGD’s ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity can be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Experiments were performed to evaluate E. coli expression strains, expression temperatures and substrate concentrations aiming for further assay optimization. Moreover, this method was evaluated on plate uniformity, signal variability and spatial uniformity. Finally, four common HGD missense variants were generated via site-directed mutagenesis and evaluated by our HTS assay.
Results
For the HTS, assay quality parameters pass the minimum acceptance criterion for Z’ values > 0.4 and SW values > 2. We found that HGD activity percentages from wildtype for each of the variants were 57.08 ± 14.42 % (for E42A), 38.56 ± 6.13 % (for M368V), 25.81 ± 7.71 % (for A122V) and 17.57 ± 6.10 % (for G161R).
Discussion and conclusion
A robust, simple, and cost-effective HTS system was developed to reliably distinguish human HGD missense variants by their HGA consumption ability. This may pave the way to the identification of novel treatment strategies for selected variants in AKU.