Deciphering Enzyme Mechanisms with Engineered Ancestors and Substrate Analogues

Environmentally friendly industrial and biotech processes greatly benefit from enzyme-based technologies. Their use is often possible only when the enzyme-catalytic mechanism is thoroughly known. Thus, atomic-level knowledge of a Michaelis enzyme-substrate complex, revealing molecular details of substrate recognition and catalytic chemistry, is crucial for understanding and then rationally extending or improving enzyme-catalyzed reactions. However, many known enzymes sample huge protein conformational space, often preventing complete structural characterization by X-ray crystallography. Moreover, using a cognate substrate is problematic since its conversion into a reaction product in the presence of the enzyme will prevent the capture of the enzyme-substrate conformation in an activated state. Here, we outlined how to deal with such obstacles, focusing on the recent discovery of a Renilla-type bioluminescence reaction mechanism facilitated by a combination of engineered ancestral enzyme and the availability of a non-oxidizable luciferin analogue. The automated ancestral sequence reconstructions using FireProtASR provided a thermostable enzyme suited for structural studies, and a stable luciferin analogue azacoelenterazine provided a structurally cognate chemical incapable of catalyzed oxidation. We suggest that an analogous strategy can be applied to various enzymes with unknown catalytic mechanisms and poor crystallizability. Many known enzymes sample huge protein conformational space, hampering structural characterization by X-ray crystallography, and preventing thus the understanding of their catalytic mechanisms. In this review, we outline that the combination of reconstructed ancestral enzymes with unconvertible substrate analogues is becoming a powerful strategy to decipher the challenging mechanisms of enzyme catalysis.image

Automated summary

Enzyme-based technologies are crucial for environmentally friendly industrial and biotech processes, but a thorough understanding of enzyme-catalytic mechanisms is necessary. This review outlines a strategy using reconstructed ancestral enzymes and unconvertible substrate analogues to decipher challenging enzyme catalysis mechanisms, which is particularly useful for enzymes with unknown catalytic mechanisms and poor crystallizability.