Chemistry Assignment | Custom Homework Help

For questions one and two, first read thesetwo literature articles (the second is cited in the first as reference 14):

Richards L, Lutz A, Chalmers DK, Jarrold A, Bowser T, Stevens GW, Gras SL. Production of metabolites of the anti-cancer drug noscapine using a P450(BM3) mutant library. Biotechnol Rep (Amst). 2019 Aug 24;24:e00372. doi: 10.1016/j.btre.2019.e00372. eCollection 2019 Dec. PubMed PMID: 31516852; PubMed Central PMCID: PMC6728265. Free full text at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728265/

Fang ZZ, Krausz KW, Li F, Cheng J, Tanaka N, Gonzalez FJ. Metabolic map and bioactivation of the anti-tumour drug noscapine. Br J Pharmacol. 2012 Nov;167(6):1271-86. doi: 10.1111/j.1476-5381.2012.02067.x. PubMed PMID: 22671862; PubMed Central PMCID: PMC3504993. Free full text at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504993/

1. Your assigned mutant for this question is VA from the Richards et al. article. Answer the following questions for that mutant.

1. What actual amino acids are different in this mutant compared to wild-type P450BM-3? What are some of the known roles (in wild-type P450BM-3) of the changed amino acids?

1. What are the major products produced by the mutant when it is given noscapine as a substrate? (Hint: Find the ‘supplementary material’ for the article for the best data.)

1. What is its ‘coupling efficiency’. What does this mean? How did the authors measure it?

1. What is the paper’s overall goal for the study, and does this mutant achieve their goal? Is it the best mutant at achieving their goal?

1. Which of the human P450s tested in the Fang et al. paper is the VA mutant most like in terms of products that it forms?

2. In the Fang et al. article, they use a ‘dual-activity’ incubation system.

1. What are the two activities, and why are both parts required to get the desired products?

1. For UGT1A1, which glucuronide metabolites (C1, C3, or C4) was it capable of producing?

1. In figure 6 panel A, it is shown that the products formed in the presence of NADPH but not GSH are different than those formed from the presence of both, and that the products formed in the presence of GSH but not NADPH are different that either of the other two sets of products. What does all of this tell us about the pathway generating the metabolites?

1. Stare in awe at Figure 8 in all its glory. What is the difference between structure I and structure X? How do compounds VIII and IX relate to each other?

For question 3, read this article:

Zhang RK, Chen K, Huang X, Wohlschlager L, Renata H, Arnold FH. Enzymatic assembly of carbon-carbon bonds via iron-catalysedsp(3) C-H functionalization. Nature. 2019 Jan;565(7737):67-72. doi: 10.1038/s41586-018-0808-5. Epub 2018 Dec 19. PubMed PMID: 30568304; PubMed Central PMCID: PMC6440214. Read free at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440214/

3. This paper (which has 160 pages of supplementary material) was submitted in July 2018, accepted in November 2018, and published in December 2018. The corresponding author, Francis Arnold, won the Nobel Prize in Chemistry for her work over the past 20 years in October 2018 while this article was being reviewed. The article describes some unusual chemistry her group made a P450 carry out, and is a good representation of the general type of work that won her a Nobel Prize.

1. How does the chemistry described in this paper differ from that of a typical P450 cytochrome?

1. They use a P450 with an unusual serine-ligated heme instead of the normal conserved cysteine. How did they arrive at the decision to use this unusual enzyme as a starting point for optimizing the activity (at least as described in the paper itself)?

1. In the paper, they refer to ‘total turnover number (TTN)’ as an important measure. What does this term actually mean?

1. Surprisingly, removal of the P450BM-3 mutant’s FAD domain resulted in higher, not lower, activity. What are two things this confirms or reveals about the chemistry?