Catalytic Efficiencies of Enolase from Fast- and Slow-Killing
Genotypes of Paenibacillus larvae
Catalytic efficiencies of enolase from fast- and slow-killing
genotypes of Paenibacillus larvaeBryan LehnerDepartment of
BiochemistryECSUHello my name is Bryan. Thank you everyone for
coming to my presentation. I hope you enjoy my presentation. I am
going to cover my thesis project which is called catalytic
efficiency of enolase from fast- and slow-killing genotypes of
Paenibacillus and really what my studying is, is a honeybee disease
I am characterizing an enzyme involved in the virulnece of a
honeybee disease. Science is important1Paenibacillus larvae causes
American Foulbrood
HoneybeeHealthy LarvaInfected with PaenibacillusBrood
combPaenibacillus larvae causes the honeybee disease American
Foulbrood and causes honeybee colony collapse by killing honeybee
larvae. If we take a snapshot into a honeybee hive we will find a
honeybee, honeybee young (honeybee larvae), and if the hive has
american foulbrood you will find borwn decayed ropy mass where a
larvae once was. This larvae has been degraded inot a brown glue
like substance by the bacterium paenibacillus. By killing honeybee
larvae, the bacteia prevents honeybee colonies from producing
enough adult honeybees to support future generations and colonies
with this disease have trouble survining.
As scinetists we want to know how this disease works and what we
have found out is that2Paenibacillus larvae kills honeybee larvae
to reproduce12345Paenibacillus larvae kills honeybees in order to
produce.
Paenibacillus is a spore forming bacterium. It produces spore
that are transmitted to honeybee hives by wind, attached to
honeybees, and on contaminated apiculture equptment. Once in the
hives the spores get mixed into the sugary sticky nectar and pollen
brood food that honeybees and honeybee larvae eat. And so, the
spores are ingested by honeybee larvae. Inside the digestive track
they germinate and massively proliferate feeding off the rich
sugary environment in the digestive tract. Because honeybee larvae
are not well developed the bacteria has the ability to degrade the
digestive tract tissue and infiltrates the circulatory fluid of the
larvae where is massively proliferates, degrades the larvae to a
foul smelling stechn , which gives american foulbrood its name, and
ultimately creates more spores that spread through the hive and to
other hives. 3Virulence varies with genotype
Slow-KillingFast-Killing 1st Genotype 2nd genotype3rd
genotype4th genotypeWe also know that not very strain is equally as
virulent. Virlence of Paenibacillus varies with genotype. There are
four genotypes of the pathogen. The 1st one kills honeybee larvae
slowly and the second, and third, and fourth kill jhoneybee larvae
quikcly. Because the slow-killing genotype is difficult for
honeybees to detect and remove from the hive the slow-killing
genotype is more virulent. It spread produces more spores inside
the hive and greater greater colony collapse. Currently the first
genotype is the second greatest threat to honeybee populations
across the world.
Because the difference in genotype has large economic and
environmental significance, we want to know what the biochemical
difference is between fast and slow killing genotypes of
Paenibacillus larvae. When I say biochemistry, we go from talking
about the organismal level to the molecular level, looking at the
machinery, that causes a difference in chemical structure and
function between these diseases. Looking at nucleic acids, the
proteins, the lipids, the individual structures that are
respinsible for this disease. And by understanidng this we can
understand the larger picture of how the disease works. 4What is
the biochemical difference between fast- and slow-killing
genotypes?Enolase is expressed in multiple places
Enolase toxic to honeybee larvae
but enolase is also an enzyme
When you start to look at what makes up a cell, you look at the
proteins that are expressed in the bacterium. Enolase is a protein
that has been found in varies places and so we started there.
Enolase is found .. And when fed.. Because it is toxic to honeybee
larvae.made up of amino acids that has been associated with
virulence of Paenibacillus. One reason is because It is found on
the surface of Paenibacillus cells, on the surface of spores. It is
found in the cytoplasm of cells. It is excreted in a suite of
virulent proteins and it has been demonstrated to be toxic to
honeybee larvae when fed to honeybee larvae in vitro. Current
studies suggest enolase plays a role in tissue degradation and has
qualities that make Paenibacillus pathogenic. However, enolase is a
multifunctional protein and as it is found in multiple plays in the
cell it is believed to have multiple functions.
Enolase is also an enzyme.
5Yes enolase might aid in tissue degradation,
but if enolase is expressed in multiple places,
maybe its enzymatic function affects virulence.
Is there a difference in catalytic efficiency between enolase
from fast and slow-killing genotypes of Paenibacillus? To test this
I am stusying to see if there is a difference between the cataytic
efficiency of enolase from fast and slow killing gentype of
Paenibacillus larvae. Does tha rate at whichc enolase carries out
its enzymatic function differ in fast and slow killing genotypes of
Paenibacillus.
6Enzymes are biological catalysts
Enzymes are biological catalysts. A catalyst accelerates the
rate at which a chemical recation occurs. There are millions of
chemical recations that occur in our body . Without enzymes these
reactions would not occur fast enough to support life. Enzymes
speed up the rate at whichc chemical recations occur, fast enough
to suppot a living system.
Which brings us to the second point. Enzymes are biological
catalysts. They are made of organic matter. Thye are proteins and
are made of eamino acids. The enzyme that I worked with look like
this and in silver you can see all the differnet loops of amino
acids. Theer are more than four-hundred amino acids in the protein,
but only a few are involved in the chemical reaction. 7Fast vs.
Slow genotypesIs there is difference in catalytic efficiency of
enolase?
Is there a difference in amino acid sequence of enolase?Cloned
enolase DNA
Fast enolaseSlow enolaseMethodsWe created enolase clones from
ERIC I Paenibacillus and ERIC III Paenibacillus. And by the blue
arrow you can see the cloned enolase gene. If you notice the strand
of DNA that the blue arrow points to is slightly higher than the
neighboring white strands of DNA, indicating that the enolase gene
has been inserted into the vecttor we used to transport the enzyme
into a bacterial cell that would produce. It9Expressed and purified
enolase protein
Fast enolase Slow enolaseWe produced and purified both enolase.
One from ERIC I and one from ERIC III. And I can prove it to you by
showing you this picture. 10Enolase converts 2-PGA to
PEP2-phosphoglycerate phosphoenolpyruvate
Rep[eated thi same reaction putitng in differnet amounts of
2-phosphoglycerateBy fitting the relation between enzyme velocity
and substrate concentration I found kinetic parametesrCalculated
catalytic efficiency
11 kcat/ Km= 2300M-1s-1
kcat/ Km = 670M-1s-1
3.4 Higher Catalytic EfficiencySlow- Killing GenotypeFast-
Killing Genotype
From Vmax I calculated the kcat the rate constant describing
ctaalytsis and with kcat and km I calculated the catalytic
efficiency.
This is important because enolase is involved in sugar
metabolism, the main metabolic pathway the bacteria relies on to
grow inside its host. Possibly the difference is virulence is due
to the fast-killing genotype can metabolize larval sugar faster,
than the slow killing genotype due to a difference in catalytic
efficiency.
12traced to a single amino acid mutation
Fast-Killing genotypeSlow-Killing genotypeDNAthat precedes the
active site!
ProteinThis mutation that is in the virulent genotype is between
the lysine and magneisum binding spots. There is a single amino
acid mutation in enolase, this mutation causes a change in
catalytic efficiency, catalytic efficiency is linked to kill=speed
of the bacterium, which is indirectly correlated to virulence,
which is repsonsible for Ameerican Foulbrood, which partcilly
responsible for honeybee population decline in the United States
over the last half century.
The mutation exists in the virulent strain. It sits adjacent to
active site. Site expected for change in catalytic
efficiency.14Fast-Killing Genotype = Higher Catalytic
EfficiencySlow-Killing Genotype = Lower Catalytic
EfficiencyPaenibacillus virulence is correlated to the catalytic
efficiency of enolaseEnolase affects ResultsPaenibacillus Life
CycleEnolase is a central enzyme in sugar metabolism and one of the
most abundantly expressed cytoplasmic enzymes in many
organisms.15
6 million2.5 millionUnited States 1940- 2010*National
Agricultural Statistics ServiceHoneybees are important pollinators
for the environment and economy. Honeybees help maintain ecosystem
stability in natural ecosystems by pollinating a wide variety of
wild flowers. Managed honeybees are important for agricultural as
they help maintain yields of nuts, fruits, and vegetables. However
since the 1940s the population of honeybees has declined by fifty
percent . In the 1940s there were six million colonies of managed
honeybees and now there are approximately 2.5 million, which has
made it more difficult for farmers to pollinate crops. This
decrease in honeybees is mainly due to disease, the main culprit
varroa mite, which is a parasitic mite that feeds on honebee
ciruclatory fluid and causes developemental defects and vectors a
variety of viruses. Beside Varroa, the main cause for honeybee
population decline is Paenibacillus larvae,
Bee pollination= 15 billion dollars of crop value (USDA
ARS)16Amino acid mutationLower Catalytic efficiency of enolase
Metabolize sugar slowerGrow SlowerKill larvae slowerEscape detected
by nurse bees Produce more spores in the hiveMore virulent to
honeybee coloniesConclusionPaenibacillus larvae kills honeybees in
order to produce.
Paenibacillus is a spore forming bacterium. It produces spore
that are transmitted to honeybee hives by wind, attached to
honeybees, and on contaminated apiculture equptment. Once in the
hives the spores get mixed into the sugary sticky nectar and pollen
brood food that honeybees and honeybee larvae eat. And so, the
spores are ingested by honeybee larvae. Inside the digestive track
they germinate and massively proliferate feeding off the rich
sugary environment in the digestive tract. Because honeybee larvae
are not well developed the bacteria has the ability to degrade the
digestive tract tissue and infiltrates the circulatory fluid of the
larvae where is massively proliferates, degrades the larvae to a
foul smelling stechn , which gives american foulbrood its name, and
ultimately creates more spores that spread through the hive and to
other hives. 17AcknowledgementsHonors ProgramCT Agricultural
Experiment StationBiology DepartmentDr. Ross KoningHoneybee
populations have declined since the 1940s
*National Agricultural Statistics ServiceAmerican Foulbrood
contributes contributes to the steady decline in managed honeybees
that has occurred since 1940. The honeybees managed for
agricultural use in the U.S. has decline by fifty percent in the
last half decade. This is largely due to varroa mite, but is
followed closely by bacterial pathogens and viruses. Paenibacillus
larvae which created a condition known as American Foulbrood is one
of the most significant other factors. Honeybee population decline
has been on the news because of colony collapse disorder, defined
by an unprecedented rate of overwinterring honeybee loss during the
winter of 2007-2008, 2008-2009, characterized by unusual symptoms.
However outside of the brief occurance, honeybee populations have
been declining due to disease, subl-lethal effects of persticides,
loss of genetic diveristy and loss of habitat. One of the main
contributers tot his steady decline that has occurred in the last
half century is Paenibacillus larvae.19Enzyme Kinetics
To determine the catalytic efficiency of enolase from fast and
slow killing genotypes of Paenibacillus larvae we determined rate
constants from a model of the enolase reaction.
Enzyme kinetics are ultimately characterizes by one value, which
is the cataltyci efficiency
20
AcidBase CatalysisAnd the important pleaces of enolase which
convert this reaction is shown in this simple diagram.
22
