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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 Valine, 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
to grow in the presence of an antibiotic (Science Daily,
2016), is one of the primary concerns that accompanies
antibiotic use. When such a bacterium carries several
antibiotic resistance genes, it is referred to as multi-drug
resistant, or a “superbug,” such as methicillin-resistant
Staphylococcus aureus (MRSA). Killing superbugs requires
new and improved antibiotics not susceptible to current
resistance mechanisms. Mannopeptimycin, a naturallyproduced antibiotic, is active against MRSA, but development
of the natural compound into a viable antibiotic has been
hampered by the fact that one of the building blocks, the
non-proteinogenic amino acid L-enduracididine, is not
available and is difficult to make synthetically. Knowing how
L-End is synthesized will aid in the development of an
inexpensive route to large quantities of L-End.
There are several residues of importance in the active site of the
MppP protein:
• Ser91, Asn160, Asp188, and Ser190 (purple) hold the PLP
cofactor and constitute the active site
• Thr12 and Glu15 (fuchsia) contact and cover the active site
• Asp227, Arg352, and Asp27 (gold) hold the L-Arg substrate (B,
light cyan) in the active site
• PLP cofactor (silver) hydroxylates Arg producing 4HKA (C, light
sky blue)
• Lys221 (lime green) is involved in the catalytic process
• Oxygen atoms are red, nitrogen atoms are blue, phosphorus
atoms are orange
Data indicate that presence of the quinoniod intermediate and
oxygen is necessary for the formation of 4HKA.
Figure 1: Antibiotic mannopeptimycin with L-enduracididine highlighted
A
II. The L-enduracididine Synthesis Pathway
The conversion of the amino acid, L-arginine (L-Arg), to the
non-traditional amino acid, L-enduracididine (L-End), is
achieved in a 3-step biosynthesis pathway. The first step (1)
in the pathway, accomplished with the enzyme MppP, adds an
oxygen atom to the L-Arg substrate, and replaces the αamino group with a ketone to create a 4-hydroxy-2ketoarginine (4HKA). The 4HKA is used in the second step
(2), the cyclization of 4HKA by MppR to give the ketone form
of End, 2-ketoenduracididine (2KE). The last step (3) is
achieved by the enzyme, MppQ, which transfers an amine
between 2KE and alanine or glycine to give L-End, which is
then incorporated into antibiotics.
1
2
B
C
Figure 3: (A) Active site of MppP from modified PDB file 5DJ3, carbon atoms are shown in various colors based on role
in the active site. (B) L-arg substrate (carbons shown in light cyan). (C) 4HKA (carbons shown in light sky blue).
IV. Enzymatic Action of MppP
MppP follows typical Michaelis-Menten kinetics where the enzyme
and substrate interact at the active site to form the enzymesubstrate complex. After reacting, the substrate has been
converted to product and leaves the enzyme intact (Figure 4a).
Through the PLP cofactor, MppP converts L-Arg and O2 to 4HKA.
• PLP bonds to the α-amino group of L-Arg
• Produces a quinonoid intermidate
• In presence of molecular oxygen, L-Arg is hydorxylated
• Produces 2 possible products (Figure 3): 4HKA and 2ketoarginine
• Supported by NMR data (Figure 4b)
3
Figure 2: L-enduracididine biosynthesis pathway
Figure 4a: This graph shows the MppP follows typical
Michaelis-Menten kinetics; The Km value for L-Arg is ~50
uM, and the turnover number (kcat is ~0.05 s-1)
Figure 4b: NMR data indicating that two products are
formed from the reaction of MppP with L-Arg and O2: 4hydroxy-2-ketoarginine (4HKA) and 2-ketoarginine
Figure 5a: UV-Visible absorption
spectra of MppP reacting with LArg over time (pink line, t=0s;
black line, t=202s)
• Figure 5a shows a peak at 510 nm that accumulates after
~60s
• Indicates the presence of the quinonoid intermediate
• 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
the enzyme.
Figure 5b: Oxygen
presence of L-Arg
consumption
in
• Figure 5B shows the amount of O2
changes upon the addition of MppP
(red arrow)
• Rate at which O2 is consumed is
dependent on the concentration of
the enzyme (1.25 uM, cyan; 2.5 uM,
light blue; 5 uM, dark blue)
• Proves the reaction with L-Arg
observed in the UV-Vis experiment
does consume molecular oxygen
• Shows oxygen is necessary for
formation of 4HKA
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
MppP-PLP dependent hydroxylation is used in subsequent
reactions in the pathway leading to the creation of the amino
acid L-End, used in some antibiotics. Easy production may help
researchers to cheaply and efficiently create new antibiotics.
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.