Functional Characterization of NUDIX Hydrolases

The NUDIX hydrolase superfamily is a diverse group of proteins consisting of approximately 50,000 known sequences found in all three domains of life: archaea, eukaryotes, and prokaryotes. NUDIX proteins catalyze the hydrolysis of NU cleoside DI phosphates linked to an X moiety by hydrolyzing the bond between sequential phosphate groups. They are commonly classified as “house cleaning” enzymes and have been identified as virulence factors in pathogens such as Legionella pneumophila . Less than 0.3% of the NUDIX proteins have been functionally characterized. Six enzymes from the NUDIX superfamily have been functionally characterized in the context of an undergraduate biochemistry teaching lab. As products of the Protein Structure Initiative (PSI), the three‐dimensional structures of these proteins have been known for many years but no functional annotation beyond the classification of “NUDIX family” has yet to be assigned. To demonstrate the importance of research‐based education in the undergraduate curriculum, we have designed and implemented a biochemistry lab as a vehicle for the improvement of undergraduate STEM education. Students are combining computational ( in silico ) and wet lab ( in vitro ) techniques as they predict enzyme function and then overexpress, purify, and characterize proteins with known structures but unknown functions. The selected enzymes represent a wide range of bacterial sources, including human pathogens. A series of assays was conducted to identify optimal reaction conditions with variables including: nine unique substrates, pHs between 7.0 – 10.5, and five different divalent metal cofactors. Each of these six enzymes demonstrated unique metal and substrate specificities and maximum activity under mildly basic conditions (pH ~8.5). The goals of this initiative are (1) to improve understanding of the process of training students to be effective scientists, (2) to test the hypothesis that “undergraduate students can characterize proteins of unknown function as the central theme of their biochemistry teaching laboratory”, and (3) to demonstrate that actually doing hypothesis‐driven experimental science is a useful tool in training students to become scientists. Our curriculum has been implemented as the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) Project on several campuses since 2016, funded in part by NSF IUSE “Collaborative Research: Using protein function prediction to promote hypothesis‐driven thinking in undergraduate biochemistry education”. Support or Funding Information NSF IUSE 1709170Structural alignment of six NUDIX hydrolases.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .