Post on 24-Feb-2016
description
M a t t h e w A uU n i v e r s i t y o f C a l i f o r n i a : M e r c e dS t a n f o r d L i n e a r A c c e l e r a t o r C e n t e rA u g u s t 1 6 , 2 0 1 2
X-Ray Crystallographic Analyses of the Antimicrobial Resistant Enzymes: β-
lactamases and Aminoglycoside Phosphotransferases
Supervised by Clyde A. Smith
Background (Infections)According to Centers for Disease Control
(CDC): Nearly 2 million patients in the U.S. get an infection in the hospital each year
About 90,000 of those patients die each year as a result of their infection, up from 13,300 patient deaths in 1992 This increase signifies that more and more bacteria are
becoming resistant to an array of treatments
Introduction In a U.S. News article about 5 months ago, doctors have
identified bacteria that produce Klebsiella pneumoniae carbapenamse (KPC) Is an enzyme that makes bacteria resistant to most known treatments , even to
the “last line of defense”: the carbapenem antibiotics Killed 50 people in Panama They have found traces in at least 37 U.S. states, Washington, D.C., and Puerto
Rico The mortality rate is at around 50% They’re most commonly found on medical equipment
Main ObjectiveHave a basis of why these two bacterial
enzymes have evolved to become resistant to these antibiotics Enzyme 1: Guiana Extended-Spectrum 1 (GES-1)
Antibiotics attached: Doripenem, Ertapenem, Meropenem
Enzyme 2: Aminoglycoside 3’-phosphotransferase (APH(2”)-IIa) and its mutant, R92H/D268N
Antibiotics attached: Gentamicin and Isepamicin
How?
X-Ray Diffraction & Crystallographyand
Structural Construction and Refinement
X- Ray DiffractionMolecules arranged in a regular, repeating pattern
in crystalsElectrons in the molecules bend an incident X-ray
beam into thousands of diffracted beamsMultiple copies of the same molecule in the same
orientation amplify the diffraction peaksThe diffraction pattern contains all of the
information necessary to determine the structure
X- Ray Diffraction Image
The data is then run through a program called HKL2000, which measures the intensity of each diffraction spot
These intensities are then converted into electron density (a probability function showing where the electrons are)
The electron density gives us a guide as to where all the protein atoms are
Structural Construction
Electron Density
3 Carbapenem Antibiotics
Carbapenem backbone
Meropenem Ertapenem Doripenem
GES-1 + Carbapenem
GES-1 + Carbapenems
GES-1 is described as a weak carbapenemase It binds the carbapenem and deactivates the antibiotic-
empowered beta-lactam ring but it doesn’t detach the drug This means the enzyme isn’t efficient for the bacteria because
eventually all the enzyme gets tied up in an inactive formHowever GES-5, an evolved form of GES-1,
deactivates and detaches the drug Leaves the active site open for infinite incoming antibiotic
carbapenems This poses a big threat for the human population
Now that we know how GES-1 binds these drugs we can start to understand how the GES enzymes are evolving into active carbapenemases
Why GES-1?
The aminoglycoside 3’-phosphotransferase [APH(2”)-IIa] enzyme is effective against an array of antibiotics: arbekacin, kanamycin, neomycin, streptomycin, gentamicin, and paromomycin
It’s drug deactivation mechanism involves the phosphorylation of the antibiotics It derives an extra phosphate group from ATP
APH(2”)-IIa Enzyme
APH(2”)-IIa + Gentamicin
A new mutant of the APH(2”)-IIa, R92H/D268N, was recently discovered to have dominance over an additional antibiotic: isepamicin Isepamicin was unable to bind to the wild-type
APH(2”)-IIa Isepamicin has a long tail which clashes with part of
the enzyme structure Asparagine 196 (Asn196)
APH(2”)-IIa Mutant
APH(2”)-IIa + Isepamicin
APH(2”)-IIa Mutant + Isepamicin
Because of the mutation at arginine 92 (it is changed to histidine), a piece of structure containing Asn196 is made more flexible Asn196 is able to bend away from the tail of the
isepamicin so that that drug can bind A phosphate group can now be attached and the drug
is deactivated
APH(2”)-IIa Mutant
Clyde Smith Stanford Linear Accelerator Center
SULI Program Coordinators at SLACOffice of Science, Department of Energy
Acknowledgements