Engineering an artificiaal metalloenzyme with a quinone coenzyme
Peter J Thompson1, Lisa Olshansky2
1Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL, United States.
None:
Artificial metalloenzymes (ArMs) have proven to be useful tools to study, modify, and exploit the chemistry at play within the active sites of naturally occurring metalloenzymes. At times, it can be challenging to study and modify naturally occurring metalloenzymes, so researchers leverage simplified scaffolds as analogues to study. Alcohol dehydrogenase enzymes that contain pyrroloquinoline quinone (PQQ) have gained more interest since 2011 as some have been found to contain a lanthanide metal within their active sites. In this chapter, we present the rationale for a PQQ containing ArM and the protocols for the expression, purification, crystallization, and characterization of this ArM as we converted a periplasmic binding protein into an ADH mimic. We envision these protocols will be useful to researchers looking to use this construct for related studies or probing other quinone-containing enzymes for similar investigation.
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