Zachary Uttke | UWM Virtual Undergraduate Research Symposium 2020

1. Characterization of an MppP
Mutant
Zach Uttke, Trevor Melkonian, Nicholas Silvaggi

2. Relevance of MppP
• MppP is involved in catalyzing the synthesis of the
nonproteinogenic amino acid L-enduracididine (L-
End).
• L-End is a building block within naturally occurring
antibiotics that have the potential to treat infections
from antibiotic-resistant pathogens.
• MppP is a very unique enzyme in that it is the first
PLP-dependent enzyme to perform a 4-electron
oxidation with just molecular oxygen and PLP, as
opposed to commonly used metals and exotic
organic cofactors.
• Furthermore, there are two groups of MppP
homologs that catalyze two different products as
shown in the image.

3. Understanding of
functional differences
• To reconcile with the difference in product
formation, a look at the active site of the enzymes is
needed. Within the active site is where differences
in structure can allude to the difference in function
between the SwMppP and the PlMppP.
• One notable difference is that SwMppP contains a
Serine residue at the 87th position, while PlMppP
contains an Alanine residue – two amino acids that
differ in both size and capability of bond formation.
This is seen in the image as an overlay between
SwMppP and PlMppP, beside the PLP cofactor.
• In addition, PLP is not convincingly seen from
electron density maps of PlMppP.
• With all this in mind, we caused an A87S missense
mutation to occur in PlMppP in hopes of seeing PLP
more securely within the active site. This structural
difference could possibly explain why the enzymes
have different activities since Serine is part of a
hydrogen bonding network that stabilizes PLP in
SwMppP.

4. Mutagenesis of PlMppP
• We used the following primers for a PCR reaction to induce the A87S missense mutation in
PlMppP. This slight mismatch of the primers allowed for the point mutation that we wanted
during PCR.
• After successfully harvesting this protein, immobilized metal affinity chromatography (IMAC)
was used to isolate pure mutant PlMppP.

5. Structure of Mutant PlMppP
• In the top image there is an overlay of the mutant PlMppP and the native
PlMppP. This shows the direct difference that the mutation caused.
• The bottom image shows just the mutant PlMppP with Serine fitting the
electron density map nicely. The density surrounding the Serine structure
is further conformation that the mutation actually occurred.
• The crystallization conditions used to order the purified mutant protein
into diffractable crystals are as shown:
• 24 mg/ml PlMppPA87S in 20mM MES pH 6.7 100uM PLP; 0.1M Bis-
Tris Propane pH 7.0 24% PEG 400 6.5% micro-seeded
• The precision values of how well the crystal was ordered are as shown:
• Resolution: 2.69 Angstroms
• R-work: 0.2050
• R-free: 0.2894

6. NMR data analyis of Native PlMppP and Mutant PlMppP
PlMppP A87S Product Analysis – 20uM
Protein + 5mM L-Arg in water
When comparing the right and middle
graphs, they are nearly identical, showing
that this mutant PlMppP appears to have
no activity. This is due to the arginine peak
being unchanged, just as in the arginine
standard.
L-Arg Standard
The right graph displays NMR peaks
showing just arginine without any catalysis
from PlMppP.
Native PlMppP Product Analysis – 20uM
Protein + 5mM L-Arg in water
This NMR data is important for showing
that PlMppP is producing dehydrated
product, as is characteristic of the 5.5
and 6.3 peaks.

7. Oxygen Probe Data
• The data is showing that each
molecule of reactant is turning
over once, sometimes fully
reacting to oxidize the arginine
and sometimes performing a half
oxidation to produce
ketoarginine.
• There are possibly trace amounts
of both the 4,5-Dehydro-
ketoarginine product and the 4-
Hydroxy-ketoarginine product.
This shows that further analysis is
needed as neither product can be
seen by the NMR
150
160
170
180
190
200
210
220
230
240
250
0 50 100 150 200 250 300
OxygenConcentration(uMol/mL)
Time (minutes)
Oxygren Probe Analysis
10uM native PlMppP with 400uM Arg
10uM A87S PlMppP with 400uM Arg
20uM A87S PlMppP with 400uM Arg

8. Conclusion
• Throughout this research project, we have been executing several
laboratory techniques to ultimately determine the activity of mutant
PlMppP. Several of the methods include:
• Designing oligonucleotide primers
• Inducing protein expression within E. coli
• Visualizing protein expression through SDS-PAGE
• Column chromatography to purify our protein
• NMR analysis of enzyme products
• Oxygen probe analysis of enzyme activity
• Crystallization of protein for X-ray diffraction
• In conclusion, it appears that the A87S mutation within the protein active
site causes oxygen turnover to occur only once. Although we are unsure of
why this is, it is reasonable to move forward with mass spectrometer
analysis to accurately determine which ketoarginine product is being made
by this mutant.