Whaddya Mean Ending Antibiotic Resistance: S. wadayamensis May Hold Answers Cudahy SMART Team: Emily Bahling, Samantha Brzezinski, Andrew Kressin, Kaylee Day, Lauren Ligocki, Katherine MacDonald, Cody Broeckel, Cori Windsor, Jason Hauk, Maddy Acherman, Madeline Romfoe, Alyssa Sims, Kaycee , Ramon Rivas, Katelyn Pomianek Advisors: Dan Koslakiewicz and Dean Billo Mentor: Nicholas Silvaggi, Ph.D.

I. Introduction III. MppP Structural Components V. Experimental Evidence for Action of MppP

The concept of antibiotic resistance, the ability of an organism There are several residues of importance in the active site of the Data indicate that presence of the quinoniod intermediate and to grow in the presence of an antibiotic (Science Daily, MppP : oxygen is necessary for the formation of 4HKA. 2016), is one of the primary concerns that accompanies • Ser91, Asn160, Asp188, and Ser190 (purple) hold the PLP antibiotic use. When such a bacterium carries several cofactor and constitute the active site antibiotic resistance genes, it is referred to as multi-drug • Thr12 and Glu15 (fuchsia) contact and cover the active site resistant, or a “superbug,” such as methicillin-resistant • Asp227, Arg352, and Asp27 (gold) hold the L-Arg substrate (B, Figure 5a: UV-Visible absorption Staphylococcus aureus (MRSA). Killing superbugs requires light cyan) in the active site spectra of MppP reacting with L- new and improved antibiotics not susceptible to current • PLP cofactor (silver) hydroxylates Arg producing 4HKA (C, light Arg over time (pink line, t=0s; resistance mechanisms. Mannopeptimycin, a naturally- sky blue) black line, t=202s) produced antibiotic, is active against MRSA, but development • Lys221 (lime green) is involved in the catalytic process of the natural compound into a viable antibiotic has been • Oxygen atoms are red, nitrogen atoms are blue, phosphorus hampered by the fact that one of the building blocks, the atoms are orange non-proteinogenic L-enduracididine, is not • Figure 5a shows a peak at 510 nm that accumulates after available and is difficult to make synthetically. Knowing how ~60s L-End is synthesized will aid in the development of an • Indicates the presence of the quinonoid intermediate inexpensive route to large quantities of L-End. • The quinonoid reacts with molecular oxygen, which is why the plot in the inset plateaus (green line) - the reaction begins to run out of oxygen • Leads to the quinonoid intermediate becoming trapped on Figure 1: Antibiotic mannopeptimycin with L-enduracididine highlighted the enzyme.

Figure 5b: Oxygen consumption in A B C presence of L-Arg Figure 3: (A) Active site of MppP from modified PDB file 5DJ3, carbon atoms are shown in various colors based on role II. The L-enduracididine Synthesis Pathway in the active site. (B) L-arg substrate (carbons shown in light cyan). (C) 4HKA (carbons shown in light sky blue). • Figure 5B shows the amount of O2 changes upon the addition of MppP The conversion of the amino acid, L- (L-Arg), to the IV. Enzymatic Action of MppP (red arrow) non-traditional amino acid, L-enduracididine (L-End), is • Rate at which O2 is consumed is achieved in a 3-step biosynthesis pathway. The first step (1) MppP follows typical Michaelis-Menten kinetics where the enzyme dependent on the concentration of in the pathway, accomplished with the enzyme MppP, adds an and substrate interact at the active site to form the enzyme- the enzyme (1.25 uM, cyan; 2.5 uM, oxygen atom to the L-Arg substrate, and replaces the α- substrate complex. After reacting, the substrate has been light blue; 5 uM, dark blue) amino group with a ketone to create a 4-hydroxy-2- converted to product and leaves the enzyme intact (Figure 4a). • Proves the reaction with L-Arg ketoarginine (4HKA). The 4HKA is used in the second step Through the PLP cofactor, MppP converts L-Arg and O2 to 4HKA. observed in the UV-Vis experiment (2), the cyclization of 4HKA by MppR to give the ketone form • PLP bonds to the α-amino group of L-Arg does consume molecular oxygen of End, 2-ketoenduracididine (2KE). The last step (3) is • Produces a quinonoid intermidate • Shows oxygen is necessary for achieved by the enzyme, MppQ, which transfers an amine • In presence of molecular oxygen, L-Arg is hydorxylated formation of 4HKA between 2KE and or to give L-End, which is • Produces 2 possible products (Figure 3): 4HKA and 2- then incorporated into antibiotics. ketoarginine • Supported by NMR data (Figure 4b) VI. Conclusions

“Superbugs” have become immune to traditional antibiotics. This has caused a need to develop new antibiotics, which can be expensive to synthesize. S. wadayamensis is capable of generating L-End, an interesting component. MppP is involved in the first step in synthesizing L-End. The 4HKA product of the 1 2 3 MppP-PLP dependent hydroxylation is used in subsequent reactions in the pathway leading to the creation of the amino

Figure 2: L-enduracididine biosynthesis pathway acid L-End, used in some antibiotics. Easy production may help Figure 4a: This graph shows the MppP follows typical Figure 4b: NMR data indicating that two products are

Michaelis-Menten kinetics; The Km value for L-Arg is ~50 formed from the reaction of MppP with L-Arg and O2: 4- researchers to cheaply and efficiently create new antibiotics. uM, and the turnover number (k is ~0.05 s-1) hydroxy-2-ketoarginine (4HKA) and 2-ketoarginine cat

References Han, L., Schwabacher, A., Graham, M., Silvaggi, N. (2015). Streptomyces wadayamensis MppP is a PLP-Dependent L-Arginine α-deaminase, γ-Hydroxylase in the Enduracididine Biosynthetic Pathway. Biochemistry 54(47): 7029-7040. Antibiotic Resistance. Retrieved February 4, 2016 from http://www.sciencedaily.com/terms/antibiotic_resistance.htm PDB File: 5DJ3 (modified)

The SMART Team Program is supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.