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Abstract
Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I. Herein, we explore ancestral sequence reconstruction as an enzyme engineering tool to enhance the therapeutic potential of PAL/TALs. We reconstructed putative ancestors from fungi and compared their catalytic activity and stability to two modern fungal PAL/TALs. Surprisingly, most putative ancestors could be expressed as functional tetramers in Escherichia coli and thus retained their ability to oligomerize. All ancestral enzymes displayed increased thermostability compared to both modern enzymes, however, the increase in thermostability was accompanied by a loss in catalytic turnover. One reconstructed ancestral enzyme in particular could be interesting for further drug development, as its ratio of specific activities is more favourable towards tyrosine and it is more thermostable than both modern enzymes. Moreover, long-term stability assessment showed that this variant retained substantially more activity after prolonged incubation at 25 °C and 37 °C, as well as an increased resistance to incubation at 60 °C. Both of these factors are indicative of an extended shelf-life of biopharmaceuticals. We believe that ancestral sequence reconstruction has potential for enhancing the properties of enzyme therapeutics, especially with respect to stability. This work further illustrates that resurrection of putative ancestral oligomeric proteins is feasible and provides insight into the extent of conservation of a functional oligomerization surface area from ancestor to modern enzyme.
Highlights
Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I
In order to enhance the therapeutic potential beyond existing PAL/TALs, in particular in terms of protein stability and relaxed substrate specificity, we turned our attention towards ancestral sequence reconstruction (ASR) as a strategy for enzyme engineering[24,25,26]
In this study we aimed to explore if ancestral sequence reconstruction could be a viable strategy to enhance therapeutic properties of phenylalanine/tyrosine ammonia-lyases from fungi, towards a complementary treatment of hereditary tyrosinemia
Summary
Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I. We believe that this PAL/TAL group harbours potential therapeutic enzymes that could be used for complementary treatment of HT1 in addition to NTBC, relieving the burden of a strict diet for patients as well as reducing accumulation of both L-Tyr and L-Phe. In order to enhance the therapeutic potential beyond existing PAL/TALs, in particular in terms of protein stability and relaxed substrate specificity, we turned our attention towards ancestral sequence reconstruction (ASR) as a strategy for enzyme engineering[24,25,26]. In our group we previously explored this approach for a terpene cyclase enzyme and obtained enzyme scaffolds with increased thermostability and promiscuity[27] Both of these properties would be of interest for a therapeutic PAL/TAL, for which prolonged half-life in the body and ability to deaminate both L-Phe and L-Tyr are of importance. Our approach would shed light on fundamental aspects of protein oligomerization that have attracted scientific interest[30,31]
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