Abstract
The suite of biological catalysts found in Nature has the potential to contribute immensely to scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with challenges. Herein we report the design of a structure-based annotation system for the identification of functionally similar enzymes from diverse sequence backgrounds. Focusing on an enzymatic activity with demonstrated synthetic and therapeutic relevance, five new phenylalanine ammonia lyase (PAL) enzymes were discovered and characterised with respect to their potential applications. The variation and novelty of various desirable traits seen in these previously uncharacterised enzymes demonstrates the importance of effective sequence annotation in unlocking the potential diversity that Nature provides in the search for tailored biological tools. This new method has commercial relevance as a strategy for assaying the ‘evolvability’ of certain enzyme features, thus streamlining and informing protein engineering efforts.
Highlights
A major challenge in the area of enzyme discovery is the accurate retrieval from databases of enzymes with specific traits, from among the vast number of related sequences, an approach hindered by misannotation from sequence curation methods which are often implemented in the absence of specialist protein functional knowledge[1]
The amine abstraction, in this case, is mediated by adduct formation with a 4-methylideneimidazole-5-one (MIO) post-translational modification in the active site[10,11]. This in turn promotes elimination in conjunction with proton abstraction via a catalytic tyrosine residue situated on an inner active site mobile loop lid[11,12]. This phenylalanine ammonia lyase (PAL)-mediated reaction often constitutes a gateway from primary metabolism to specialist compound biosynthesis, with examples including phenylpropanoids in plants[13] and various antibiotics in bacteria (Fig. 1a)[14,15,16,17]
One such example is phenylketonuria, an inborn error of metabolism in which metabolic dysregulation leads to neurological symptoms such as microcephaly, seizures and intellectual disabilities[18]
Summary
Several crystal structures of PAL enzymes have been solved, none exists with the natural ligand trans-cinnamic acid bound in the active site, thereby preventing correct and unambiguous assignment of the phenylalanine-specific MIO-dependent ammonia lyase zymophore. Other differences included H359 moving into the active site sphere in the cinnamate bound structure, along with smaller shifts in the positions of Q311 and R317 (SI, Fig. S1) These represent movements which occur upon substrate binding, they may be induced instead by the Y78F variation as exchange of polar and non-polar residues in the inner active site loop of related enzymes has been reported to influence conformational dynamics[39]. These three seemingly mobile positions have been identified as hotspots in mutagenic studies of various PAL-relatives, with variation at H359 shown to affect catalytic efficiency[36] and the two carboxylate-binding positions (Q311 and R317) being implemented in substrate positioning and selectivity[40,41].
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