YZGD, a Two‐Domain Enzyme, as a Teaching Tool in a Biochemistry Laboratory

Abstract

YZGD is a two domain enzyme that is both a member of the HAD superfamily with pyridoxal phosphatase activity and a member of the Nudix Hydrolase superfamily with comparable activity on CDP alcohols, ADP sugars and ADP coenzymes, TDP sugars, and some activity on UDP and GDP sugars. Due to its two‐domain nature, I have found it to be a useful enzyme to use in the teaching laboratory. We have created a mutant (YZGDD12N) that has no pyridoxal phosphatase activity but full Nudix activity and a mutant (YZGDE324Q) that has no Nudix activity but full pyridoxal phosphatase activity, demonstrating the independent nature of enzyme domains. We also have cloned both a his‐tagged YZGD and a non‐his‐tagged YZGD, thus demonstrating the power of affinity chromatography in enzyme purification. The students express the various YZGDs (mutants, wildtype, and his‐tagged), purify these enzymes, and characterize them (Bradford assays, gel electrophoresis, and enzyme assays). We then work as a group to compare and contrast the results that each student gets; it is this comparison that especially shows the students the power of site‐directed mutagenesis and affinity chromatography purification. In addition, the students do a protein structure ‐ function project where they use the structural database SCOP, the visualization program CN3D, BLAST searches and CLUSTAL alignments, and the scientific literature on any HAD or Nudix superfamily member that has been structurally determined.