Article Information: PMID: 30023842 InternalPaperNumber: Luc-C5 Protein: FLuc ReviewOrPrimaryArticle: Primary FirstAuthor: Pozzo, Tania CorrespondingAuthor: Yokobayashi Journal: ACS Year: 2018

MutantName: YY5 Mutation(s): I423L, D436G, L530R, T214A, A215L, I232A, F295L, and E345K Substrate(s): D-luc FunctionChange(s): YY5 displays a red-shift in the emission spectrum. YY5 also exhibits similar stability characteristics as Mutant E (60% activity at 45ºC), except has slower decay rates at 35ºC-45ºC. This is significantly more stable than WT, which loses 70% activity at 35ºC and inactive at 45ºC. YY5 has a higher Km than FLuc (lower than Mutant E), but highest Kcat out of the other enzymes tested (FLuc, LGR, and Mutant E). BiochemicalHypotheses: Since most of the LGR mutations are located in the CTD and most of Mutant E mutations are located in the NTD, it is suggested that the two sets of mutations do not interfere with the phenotypes in YY5, contributing to the increased thermostability and enhanced affintiy. UsefulReferences:

Article Information: PMID: 30023842 InternalPaperNumber: Luc-C5 Protein: FLuc ReviewOrPrimaryArticle: Primary FirstAuthor: Pozzo, Tania CorrespondingAuthor: Yokobayashi Journal: ACS Year: 2018

MutantName: Mutant E Mutation(s): T214A, A215L, I232A, F295L, and E345K Substrate(s): D-luc FunctionChange(s): Increased thermostability compared to FLuc. BiochemicalHypotheses: UsefulReferences: [Baggett, B.; Roy, R.; Momen, S.; Morgan, S.; Tisi, L.; Morse, D.; Gillies, R. J. Thermostability of firefly luciferases affects efficiency of detection by in vivo bioluminescence. Mol. Imaging 2004, 3, 324– 332, DOI: 10.1162/1535350042973553]: First to construct the mutant and determine the increased thermostability. [ Branchini, B. R.; Ablamsky, D. M.; Murtiashaw, M. H.; Uzasci, L.; Fraga, H.; Southworth, T. L. Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications. Anal. Biochem. 2007, 361, 253– 262, DOI: 10.1016/j.ab.2006.10.043]: Determined that combining these 5 point mutations with other mutants increased the stability of the enzyme.