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