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Transcript
X-Ray Crystallographic Analyses of the
Antimicrobial Resistant Enzymes: βlactamases and Aminoglycoside
Phosphotransferases
Matthew Au
University of California: Merced
Stanford Linear Accelerator Center
August 16, 2012
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 Objective
 Have 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 & Crystallography
and
 Structural Construction and
Refinement
X- Ray Diffraction
 Molecules arranged in a regular, repeating pattern in
crystals
 Electrons in the molecules bend an incident X-ray beam
into thousands of diffracted beams
 Multiple copies of the same molecule in the same
orientation amplify the diffraction peaks
 The diffraction pattern contains all of the information
necessary to determine the structure
X- Ray Diffraction Image
Structural Construction
 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
Electron Density
3 Carbapenem Antibiotics
Carbapenem backbone
Meropenem
Ertapenem
Doripenem
GES-1 + Carbapenem
GES-1 + Carbapenems
Why GES-1?
 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 form
 However 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
APH(2”)-IIa Enzyme
 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 + Gentamicin
APH(2”)-IIa Mutant
 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 + Isepamicin
APH(2”)-IIa Mutant + Isepamicin
APH(2”)-IIa Mutant
 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
Acknowledgements
 Clyde Smith
 Stanford Linear Accelerator Center
 SULI Program Coordinators at SLAC
 Office of Science, Department of Energy