recombinant expression, purification and kinetic
TRANSCRIPT
RECOMBINANT EXPRESSION, PURIFICATION AND KINETIC CHARACTERIZATION OF
F420-COFACTOR DEPENDENT GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM
MYCOBACTERIUM TUBERCULOSIS
by
TIJANI OSUMAH
Presented to the Faculty of the Graduate School of
The University of Texas at Arlington in Partial Fulfillment
of the Requirements
for the Degree of
MASTER OF SCIENCE IN BIOENGINEERING
THE UNIVERSITY OF TEXAS AT ARLINGTON
May 2013
ii
Acknowledgements
My mother always said to put God first. So I would like to thank God first for all He has
done for me. Without Him, I would not be who or where I am at this moment.
Huge thanks go to my mother, Mrs. Zulietu Osumah, father, Dr. Aruna Osumah and
other mother, Mrs. Awawu Osumah, for their unending love, prayers and support. Also, I would
like to say a big thanks to my many brothers and sisters. I grew up watching all of you. I lived
and learned from your mistakes. I kiss the ground you all walk on. All that I am is because of
you. Thank you so much.
I would also like to extend my unending gratitude to my P.I., Dr. Kayunta Johnson-
Winters. Dr. Kay, you have been like a mother to me. I pray for our long lives, so that I can be
like a son to you.
Last but not least, a loving thanks to my adopted cousin, Adebanke Adetola and to all of
my friends who’ve been with me through it all. I look forward to all the greatness you will all
accomplish. Best wishes, love and respect.
April 18, 2013
iii
Abstract
RECOMBINANT EXPRESSION, PURIFICATION AND KINETIC CHARACTERIZATION OF F420-
COFACTOR DEPENDENT GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM
MYCOBACTERIUM TUBERCULOSIS
Tijani Osumah, MS
The University of Texas at Arlington, 2013
Supervising Professor: Kayunta Johnson-Winters
The FGD (F420-dependent Glucose-6-phosphate dehydrogenase) enzyme is an F420
Cofactor (7,8-didemethyl-8-hydroxy-5-deazariboflavin) dependent enzyme found in
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). TB is still a prominent
cause of illness and death worldwide. Because FGD is not found in humans, makes it a good
target for drug development. By understanding the hydride transfer mechanism of the FGD in
detail, we can aid in the improvement of drug targeting and development for the treatment of TB.
FGD catalyzes the conversion of glucose-6-phosphate to 6-phosophogluconolactone.
This project focuses on the purification and kinetic characterization of recombinant FGD using
steady-state and pre-steady state kinetic methods. A concurrent goal is to probe the functionality
of conserved active site residues that are involved in the hydride transfer reaction. Based upon
crystallographic data, it is believed that Histidine 40 acts as an active site base, abstracting a
proton from the substrate, glucose-6-phosphate, facilitating the hydride transfer from the
substrate to the F420 Cofactor. A separate active site amino acid, Tryptophan 44 is believed to
stabilize an active site intermediate during turnover. We have mutated these conserved
residues, making the following FGD variants, H40A, W44F and W44A. Here, we present
purification methods and steady-state kinetic analyses of both wild type FGD and the H40A
variant.
iv
Table of Contents
Abstract ......................................................................................................................... iii
List of Illustrations ........................................................................................................... v
List of Tables ................................................................................................................. vi
Chapter 1 Introduction .................................................................................................... 1
1.1 Phylogenetic distribution of the F420 Cofactor ................................................. 5
1.2 Enzymes: Relevance of the cofactor through enzymes .................................14
Chapter 2 F420-Dependent Glucose-6-Phosphate Dehydrogenase (FGD) ......................16
2.1 Expression and Purification of FGD ...............................................................20
2.1.1 Expression of cell extracts ..............................................................20
2.1.2 Purification of FGD .........................................................................20
2.1.3 Site-directed mutagenesis of active site residues in FGD ...............22
2.1.4 Crystallography ..............................................................................24
Chapter 3 Results .........................................................................................................26
3.1 Purification of wtFGD and mutant variants ......................................................26
3.2 Active site mutations .......................................................................................27
3.3 Steady-state kinetics and pH profile ................................................................27
3.4 Conclusions ....................................................................................................30
References ...................................................................................................................33
Biographical Information ................................................................................................38
v
List of Illustrations
Figure Page 1-1 F420-1 Cofactor and its smaller fragments (FO and F+) ........................................... 3 1-2 Spectra of the oxidized and reduced F420 Cofactor.................................................. 4 1-3 Ionization and resonance structure of F420 Cofactor ................................................ 5 1-4 Phylogenetic tree of the archaea ............................................................................ 7 1-5 Multiple sequence alignment of the FbiC protein..................................................... 10 1-6 Phylogenetic relationship between the PPOx, FDR-A, FDR-B families .................... 13 1-7 Average numbers of putative F420/FMN-binding protein family genes in actinobacterial species ....................................................................................... 14 2-1 Catalyzed reaction of FGD ...................................................................................... 17 2-2 Nitroimidazopyran pro-drug PA-824 ........................................................................ 17 2-3 Crystal structure of FGD ......................................................................................... 19 2-4 Proposed mechanism of FGD ................................................................................. 19 2-5 Schematic of protein purification procedures ........................................................... 22 2-6 Sequence chromatograms showing successful mutation ......................................... 24 2-7 Crystals formed from varying crystallization conditions of the H40A variant ................................................................................ 25 3-1 SDS-PAGE 1 .......................................................................................................... 26 3-2 SDS-PAGE 2 .......................................................................................................... 27 3-3 Steady state kinetic plot .......................................................................................... 29 3-4 pH profile of wtFGD and H40A variant in 50mM K3PO4 at 25°C .............................. 30 3-5 Distance of the histidine amino acid residues in the active site from the modeled substrate .................................................................................... 30
vi
List of Tables
Table Page 1-1 Distribution of 7,8-didemethyl-8-hydroxy-5-deazariboflavin ..................................... 6
3-1 Summary of concentration and volume parameters of kinetic assays ...................... 28
3-2 Steady-state kinetic assay parameters of wtFGD and
H40A FGD mutant variant. ...................................................................................... 28
1
CHAPTER 1
Introduction
The availability of an unusual electron-transfer coenzyme, known as the F420 Cofactor
(a 7,8-didemethyl-8-hydroxy-5-deazaflavin transferring agent) (Figure 1-1) has been understood
to be vital for catalysis in certain enzymes. The F420 Cofactor is chemically equivalent to NAD+,
which is exclusively involved in hydride transfer reactions (Equations 1 and 2). However, the
chemical structure is reminiscent of an isoalloxazine chromophore with a side chain comprising
of ribitol, phosphate, and lactate residues as well as a ɣ-linked polyglutamate tail that varies in
length (F420-1 to F420-9), depending upon the species in which the coenzyme is found 5.
(Equation 1)
(Equation 2)
The F420 Cofactor can be oxidized or reduced and is therefore, spectrophotometrically
distinct (Figure 1-2). In a basic solution, the Cofactor is oxidized and bright yellow in color, with
an absorbance maximum between 401-420 nm (Figure 1-2) 6-7
. The oxidized cofactor is an
obligate two-electron acceptor under physiological non-photoreductive conditions 6. Upon
reduction, it loses the 420 nm absorbance and gains a new absorbance at 320 nm 8 with a
much lower extinction coefficient (at pH 8.85, ε420 of F420 is 45,500 M-1
cm-1
and at pH 8.8, ε320
of F420 H2 is 10,800 M-1
cm-1
) 9-10
. Below pH 6.0, the maximum absorbance of the oxidized
Cofactor shifts from 420 nm towards lower wavelengths9-10
. The isosbestic point between pH 4-
10 has been previously determined to be 401 nm. However, as the pH is increased, the
hydroxyl proton at the 8 position (pKa = 6.3) 9 of the F420 Cofactor (Figure 1-3; structure A) is
removed, consequently converting the F420 Cofactor into an anionic form, which has two
resonance structures (Figure 1-3; structure B and C) 10
. These resonance structures attribute for
the shifting of the absorbance wavelength 10
. Also, at a basic pH, another proton is removed
from the 3-NH (pKa = 12.2), resulting in the formation of structure D (Figure 1-3), this also
results in absorbance shifts to longer wavelengths 10
. Similar to the chemical structure, the
2
fluorescence spectral properties of the F420 Cofactor are also pH and redox sensitive 8, 10
. The
oxidized cofactor has strong fluorescence at 425 nm, while the reduced form does not. FO and
F+ (Figure 1-1), two of several acid-hydrolysis products of the F420
Cofactor, exhibit spectral
properties that are similar to those of the unaltered F420 Cofactor. However, these hydrolysis
products have an extinction coefficient at 420 nm that is approximately 15% less than that
exhibited by the F420 Cofactor. Also, FO and F+ have been observed to possess a shorter
nitrogen-10 side chain than that of the F420 Cofactor. Nonetheless, both FO and F+
compounds
are catalytically active in several F420-dependent enzymatic reactions 9-10
. Additionally, the F420
Cofactor is photosensitive, rapid decomposition occurs when it is exposed to a strong white
light, especially at basic pH 11
.
The redox potential of the F420 Cofactor is between -340 to -350 mV (much lower than
that of flavins) 12
. This lower redox potential has been attributed with several major cellular
implications. For example, instead of mediating the transfer of electrons between NAD+ and
higher potential one- and two-electron acceptors such as flavins, the F420 Cofactor has the
capacity to mediate the reduction of NAD+ with electrons from hydrogen or via formate oxidation
13. In comparison to flavins, which have accessible semiquinone, the absence of the
semiquinone or its formation has no deleterious effects on this role.
Nonetheless, the F420 Cofactor serves as a useful biochemical tool in the studies of
flavoprotein catalysis. It is stable to air and boiling at near neutral pH, degraded by light at high
pH and the side chain is cleaved in a low pH environment. It remains unknown whether, except
in the photolyase, the F420 Cofactor is ever enzyme bound. It appears to behave as a soluble
electron-transfer cofactor, but it has not been thoroughly investigated for its ability to covalently
bind to proteins 6.
3
Figure 1-1 F420-1 Cofactor and its smaller fragments (FO and F+). The only difference is the length of the side chain
17
NH
NNHO
HO
OH
HO
O
O
O
P
OO
O-
O
HN
5
10
FO
F+
F420-0
COO-
OH
O
F420-1
4
Figure 1-2. Spectra of the oxidized and reduced F420 Cofactor along with corresponding structures (A. oxidized F420, B. reduced F420 :H2 Cofactor). Data are from Tzeng and other
coworkers 65
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
300 400 500 600
Ab
so
rba
nc
e
Wavelength (nm)
A) Oxidized F420 Cofactor
B) Reduced F420 Cofactor
5
Figure 1-3. Ionization and resonance structure of F420 Cofactor
10: Removing a proton of the 8-
OH group (pKa = 6.3) 9 of the F420 Cofactor (Structure A) converts F420 into an anionic form, which has two resonance forms (Structure B and C)
10
1.1 Phylogenetic Distribution of the F420 Cofactor:
The infrequent presence of the F420 Cofactor and its biosynthesis in certain prokaryotes has
been utilized in comparative phylogenetic analysis and genomics-based investigation of
organisms that employ this cofactor in their metabolic pathways. The F420 Cofactor has been
previously discovered to be distributed sporadically amongst certain prokaryotic organisms, but
observed universally in mycobacteria 14
. However, before this discovery, the F420 Cofactor was
known to play an extensively significant role only in methanogenic bacteria and archaea. While
this distribution is wide-ranging, the F420 Cofactor is abundant only in methanogenic bacteria
where it is believed to function primarily as a redox carrier in energy metabolic pathways.
Although the cofactor is not unique to methanogens, it has become an important indicator for
the identification of methanogens because of its high abundance in these cells and its intense
6
fluorescence, especially when compared to other species in which this cofactor is found (Table
1)14a, 15
.
Table 1-1 Distribution of 7,8-didemethyl-8-hydroxy-5-deazariboflavin 15a
Organism Content expressed in pmol/mg (dry weight)
Methanogenic archaebacteria
-Methanobacterium thermoautotrophicum 3800
-Methanospirillum hungatei 3700
-Methanobacterium formicium 2400
-Methanosarcina barkeri 190
Nonmethanogenic archaebacteria
-Halobacteria
Strain GN-1 >210
Halococcus salinarium >33
-Acidophilic archaebacteria
Thermoplasma strain 122-1B3 >5
Sulfolobus solfataricus >1
Eubacteria
Streptomyces spp. <20
Mycobacterium tuberculosis 13.5
Nocardia aurantia 8.5
Anacystus nidulans NR
In fact, the F420 Cofactor was found in all methanogens examined at levels varying from
1.2 mg/kg of cell dry weight as in the case of Methanobervibacter ruminatium to approximately
65 mg/kg of cell dry weight as seen in Methanobacterium thermoautotrophicum 16
. All
methanogenic bacteria subsequently discovered have been observed to possess genes that
7
encode for the synthesis, and ultimately the utilization of the F420 Cofactor in one way or another
16-17. Because of its abundance, this deazaflavin Cofactor apparently plays a critical role in the
physiology of methanogens. Several authors have postulated that the F420 Cofactor is a
functional substitute for ferredoxin16
. This idea has been supported by the absence of ferredoxin
in several methanogens that have been studied. Also, an investigation of Methanosarcina
barkeri found that it possessed an F420 Cofactor with polyglutamate derivatives at an order of
magnitude much lower than in other methanogens, but contained ferredoxin 15a
. Most
biochemical and energetic studies have been performed with a few methanogenic genera:
Methanosarcina, Methanobacterium and Methanococcus. Since these genera are
phylogenetically distantly related (Figure 1-4), the results obtained were hence considered to be
representative for all methanogens6, 15b
.
Figure 1-4. Phylogenetic tree of the archaea indicating the phylogenetic relationship between various methanogenic genera, Archaeoglobus and Pyrococcus
6.
Also, the F420 Cofactor is found in Archaeoglobus fulgidus, where it participates in a
series of two-electron transfer reactions. A. fulgidus is the first sulfur metabolizing organism to
have its genome sequence determined. It possesses all of the enzymes and cofactor required
for methanogenesis and produces a measurable amount of methane during sulfate reduction.
8
Levels of the F420 Cofactor found in A. fulgidus are similar to methanogens 6, 18
, the discovery of
this cofactor in other organisms suggests that the chemistry of the compound has been useful in
other areas of cellular metabolism.
Although the structure of the F420 was initially determined from methanogenic bacteria19
,
it was present in all Streptomyces species examined, as well as a pool of other related
organisms such as Nocardia6,15a,17,20
. It is also present in one genus of eukarya, and in some
other archaea. Analysis of Streptomyces showed that some F420 is excreted by the organism
during growth; however, it is released in the form of FO. More so, compared to methanogens,
the levels of the F420 Cofactor in Streptomyces was found to be about 10-fold lower. The role of
the F420 Cofactor in most Streptomyces is not known, but in specific Streptomyces, it is vital for
the synthesis of chlorotetracycline, oxytetracycline, and lincolnomycin, as well as DNA light
repair 17, 20-21
. Also, the biosynthesis of riboflavin in Methanobacterium thermoautotrophicum
was studied by Eisenrich et al. (1991) and was found to be identical to that in eubacteria and
fungi 15b, 22
. In the green algae Scenedesmus, the deazaflavin ring of the F420 Cofactor is
required for DNA photolyase function14a, 15a, 21
.
In 2002, the F420 Cofactor became popular as investigation into its function and
mechanism in mycobacteria amassed rapid attention. Purwartini and Daniels have shown that
the F420 Cofactor is involved in the oxidation of glucose-6-phosphate dehydrogenase by and
F420 dependent glucose-6-phosphate dehydrogenase (FGD1, Rv0407 – MTB gene). By doing
so, the F420 Cofactor is biochemically modified to its reduced form 14a
. Phylogenetic investigation
of F420 biosynthesis, using M. bovis as a model, were also studied. Choi et al (2001, 2002)
showed that fbiC gene participates in the earlier steps of F420 biosynthesis between
pyrimidinedione and hydroxyphenyl pyruvate to form FO by encoding for synthase. fbiAB genes
are responsible for the biosynthesis of F420 from the precursor, which involves the addition of a
phospho-lactate group and condensation of glutamate on . M. tuberculosis, M. bovis, M. avium,
9
M. leprae, Nocardia farcinica, Streptomyces coelicolor, S. avermitilis, Thermobifida fusca and
Rubrobacter xylanophilus all have proteins with high homology for full length fbiC as shown in
multiple amino acid sequence alignment of fbiC using representative organisms (Figure 1-5). In
contrast, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, Methanococcus
jannaschii, Halobacterium sp., Synechocystis sp., and Nostoc sp. all have two polypeptides
(located adjacent or non-adjacent) which encode for fbiC23
.
10
Figure 1-5. Multiple sequence alignment of the FbiC protein in Mycobacterium sp., Norcadia sp. and Streptomyces sp.
23c.
11
Selengut and Haft (2010) implemented partial phylogenetic profiling (PPP) to discover
protein families that utilize the F420 Cofactor 14b
. This method was efficient in determining these
protein families due to the possibility that the distribution of the F420 Cofactor may not span the
entire profile. By applying hidden Markov models (HMMs) to a set of all 1,451 bacterial and
archaeal genomes available from the National Center for Biotechnology Information (NCBI),
several F420-utilizing enzymes in mycobacteria based on the pattern of the F420 Cofactor
biosynthesis trait were identified. The most prominent family in the distribution was the
Luciferase-like Monooxygenase (LLM) family. A few LLM family proteins have previously been
discovered in archaea to be F420 dependent; one has even been characterized 14b, 24
.
The second most prominent family of enzymes from the partial phylogenetic profiling
was the deazaflavin-dependent nitroreductase (DDN) families. This family was determined to be
limited to only F420-producing species. Like the LLM family, the only characterized member of
this family is F420 dependent. The DDN family is known to include the deazaflavin-dependent
nitroreductase Rv3547, this protein is active in the activation of the promising antimycobacterial
prodrug, PA-824 14, 23a, 23c, 25
.
The third family discovered from this profiling was the pyridoxamine 5’-phosphate
oxidase (PPOX, or PNPOx). At the time of this discovery, there were no known F420-dependent
members of the PPOx family, however, several FMN-dependent enzymes were known. More
recently, two families of F420 dependent reductases (FDR-A and –B) which were previously
uncharacterized have been identified. These enzymes were discovered to be distantly related to
PPOXs (Figure 1-6). However, the PPOx and FDR families are functionally dissimilar.
Structurally and phylogenetically, the FDRs and PPOxs appear to have a common evolutionary
origin, yet these enzyme families have evolved to utilize different Cofactors. Irrespective of their
structural similarities, there are at least two major chemical differences between the two
Cofactors. As a result, catalyzed reaction by the two enzyme families is opposite (oxidation by
12
the PNPOxs versus reduction by the FDRs). Taylor et al. suggests that this difference in
catalytic activity resulted in the evolutionary expansion of the FDRs in M. smegmatis (28 genes)
compared with the single PPOx gene. Due to the high reducing power of F420, these enzymes
have hence been allowed to catalyze the reduction of a wide variety of compounds, particularly
xenobiotics14b, 26, 27
.
These three (LLM, DDN and PPOx) families account for 32 genes in Mycobacterium
tuberculosis (Figure 1-7) and 123 genes in Mycobacterium smegmatis. Partial phylogenetic
profiling was also able to identify other members of LLM and PPOx as F420-Cofactor related,
however, it was unable to determine how many and which ones specifically amongst the
uncharacterized actually bind to the cofactor 14b
.
13
Figure 1-6. Phylogenetic relationship between the PPOx, FDR-A, FDR-B families 26
.
14
Figure 1-7. Average numbers of putative F420/FMN-binding protein family genes in actinobacterial species
30.
1.2 Enzymes: Relevance of Cofactor Through Enzymes
The F420 Cofactor was first found in methanogenic bacteria by Wolfe and his co-workers
31,32. It was initially thought to be a unique cofactor to methanogens
33, but was later discovered
to be present in several other organisms including Streptomyces 5,34-35
, Mycobacteria 36-37
,
Cyanobacteria 38-40
, green algae 41
and halobacteria 10
. Methanogens play a critical role in
carbon cycling because the methanogenic pathway reduces carbon dioxide to produce methane
(Equation 3).
15
(Equation 3)
As mentioned previously, several F420 Cofactor dependent enzymes have been
identified and observed to require this cofactor for catalysis (Table 2) 8. The most important
component of the F420 Cofactor needed for catalytic activity is the isoalloaxine chromophore 42
(Figures 1 & 2). The consequence of the carbon substitution at the 5-position is the conversion
of the central ring from a pyrazine to a pyrimidine. This change significantly modifies the
thermodynamics and kinetics of the redox processes involved with this Cofactor. Because of
this change, 5-deazaflavins, like the F420 Cofactor, are restricted to the two electron transfer
cycles and only wield half the versatility of flavins. As a result, they are reduced more slowly
when constituted with apoflavoenzymes, but reduced rapidly when the specific oxidant is a two-
electron acceptor 43
.
The alteration of other portions of the F420 Cofactor has also been observed to have a
significant effect on F420 flavoenzyme kinetics. For example, it has been shown that changing
the length of the side chain at nitrogen 10 (as in FO and F+) alters the rates of enzymatic
reactions or the Km for the substrate, but the nature of catalysis remains the same 42
. The F420
reducing hydrogenase in cell extracts of Methanobacterium strain M.o.H has an apparent Km of
25 µM for the cofactor, however, it exhibits a higher Km of 100 µM for F+ and FO compound
33.
More so, for the F420-specific secondary alcohol dehydrogenase, the hydroxyl at Carbon 8 (C-8)
position influences the spectroscopic properties of the F420 Cofactor and the pH activity profiles
of the enzyme. Furthermore, methylation at C-8, C-7, or C-5 reduces the catalytic activity of the
F420 Cofactor, and an alteration of the ring structure inactivates the cofactor 42
.
16
CHAPTER 2
F420-Dependent Glucose-6-Phosphate Dehydrogenase (FGD)
Unlike most of the previously discussed enzymes, F420 Dependent Glucose-6-
Phosphate Dehydrogenase (FGD) plays no role in methanogenensis or sulfite reduction. FGD
has been discovered in Mycobacteria and Norcadia species, but, its crystal structure has only
been solved from Mycobacterium tuberculosis (Mtb). FGD catalyzes the conversion of glucose-
6-phosphate (G-6-P) to 6-phosphogluconolactone (Figure 2-1, Equation 10). This reaction is a
vital metabolic step in survival of the species in which this enzyme is present 2,24
.
(Equation 10)
Similar to every F420 dependent enzyme, the reduction of the F420 Cofactor is achieved
by the hydride transfer from the substrate molecule (G-6-P) to the C5 position of the cofactor
ring (Figure 2-1). However, the role of the F420 Cofactor to Mtb in general has not been fully
investigated nor understood.
Nonetheless, the F420 Cofactor has been discovered to have some therapeutic
significance. PA-824 (Figure 2-2) is a nitroimidazopyran pro-drug, which was synthesized in the
efforts of combating the tuberculosis pandemic 24
. Tuberculosis remains a major cause of illness
and death worldwide (Center for Disease Control Data, 2011). It is estimated that one third of
the world’s population (approximately 2 billion people) carry this organism and that there are
about 9 million new cases per year occurring at a rate of about one per second. In 2011, there
were 1.4 million TB-related deaths worldwide.
17
Figure 2-1. Catalyzed reaction of FGD coupled with the activation of anti-tubercular pro-drug PA-824
Figure 2-2. Nitroimidazopyran pro-drug PA-824
Probing the mechanistic interactions between the pro-drug and the F420 Cofactor, it was
discovered that the cofactor is indirectly responsible for the activation of the pro-drug. The
N
N
O
O
O2N
OCF3
Figure 3: Strucure of PA-824 drug
18
studies showed that the reaction which FGD catalyzes produces reduced F420 to an accessory
protein (Rv3547) which in turn activates PA-824 14a,17,24,43
. The research goal of this project is to
understand the hydride transfer reaction mechanism of FGD in detail. One avenue to study this
reaction is to use steady-state and pre-steady state kinetic methods. Additionally, hydride
tunneling within FGD will be investigated using kinetic isotope effects (KIE). However, this goal
is beyond the scope of this work. More so, site-directed mutagenesis will be used in order to
understand the function of specific residues within the active site of FGD that are believed to
interact with the substrate and intermediates during catalysis.
Before any such studies can be conducted, wild type FGD (wtFGD) must first be
expressed, purified and then characterized. Therefore, the initial emphasis of this project was
the optimization of the expression and purification of wtFGD. A concurrent goal was to mutate a
conserved Histidine residue within the active site of FGD. The crystal structure of FGD from M.
tuberculosis has already been solved using multiwavelength anomalous dispersion methods.
The structure was determined to be an α/β triosephosphateisomerase (TIM) barrel homodimer
(Figure 2-3) with a molecular weight of 78 kDa. Each monomer was determined to weigh
approximately 47 kDa. Based upon this crystal structure, it was postulated that an active site
Histidine (His 40) acts as an active site base which abstracts a proton from the O1 hydroxyl of
glucose-6-phosphate (Figure 2-4). This abstraction facilitates the hydride transfer within FGD.
The reaction continues with the hydride transfer step from the C1 of G-6-P to the C5 of F420.
Finally, the electronic rearrangements in the isoalloxazine system that accompany reduction
also require protonation at N2 in the pyrimidine ring by Glu 109 24
.
To fully understand the functionality of this residue, the H40 amino acid residue was
mutated to an alanine (His40Ala or H40A). This will provide insight into the hydride transfer
reaction mechanism that occurs within FGD as well as the interaction between G-6-P and other
amino acid residues within close proximity. Both projects are consequently described.
19
Figure 2-3. Crystal structure of FGD, with the F420 Cofactor (green) buried within the active site
24
Figure 2-4. Proposed mechanism of the FGD enzyme
24
20
2.1 Expression and Purification of FGD
As previously stated, the initial goal of this project is to express and purify recombinant
FGD from Mycobacteria tuberculosis. Because M. tuberculosis is an air borne pathogen, it was
unfavorable to undertake our studies on the FGD enzyme from its native source. Therefore, the
gene has been subcloned into an expression vector, pBluescipt (Stratagene, Santa Clara, CA).
pBluescript is compatible with expression in BL21(DE3) E. coli cells which are non-pathogenic.
A purification protocol has been established previously with recombinant FGD from M.
smegmatis14a
. Because of the high sequence homology between FGD from M. tuberculosis and
FGD from M. smegmatis, it was reasonable to suggest that the purification protocols would be
similar. However, this was not the case. A novel and effective purification protocol for FGD was
developed for the studies outlined in this work.
2.1.1 Expression of cell extracts:
Aliquots from frozen BL21(DE3) E.coli cell stocks were plated on LB agar, containing
50 mg/mL Ampicillin. After incubation for 9-12 hours at 37°C, a single colony from the plates
was used to inoculate 500-1000 mL of Luria-Bertani (LB) broth containing 50 mg/mL Ampicillin.
The cell culture was used to inoculate 10 liters of LB broth containing 50 mg/mL Ampicillin. The
cells were grown at 37°C until an OD600 of 1.00 was attained. Next, IPTG (isopropyl β-D-1-
thiogalactopyranoside) was added to a final concentration of 0.3 mM as an inducer. The cells
were incubated at 37°C for an additional 15 hours for expression of the FGD enzyme.
2.1.2 Purification of FGD:
Unless otherwise stated, all subsequent purification procedures were undertaken at
4°C. Harvested cells were resuspended in 20 mM Tris-HCl buffer (pH 7.0) and lysed via
sonication using a Branson Sonicator (Branson Ultrasonic Corporation, Danbury, CT) three
times at 50% amplitude for 3 to 4 minutes each time, while maintaining the temperature below
10°C. The resulting cell lysate was centrifuged at 12,000 rpm for 30 minutes at 4°C.
Streptomycin sulfate (Figure 2-5) was then added to the supernatant to a final concentration
21
of 1.5% w/v. The resulting sample was then stirred using a stir-bar for 10 minutes and then
centrifuged at 11,000 × g for 20 minutes.
The resulting supernatant (post-streptomycin sulfate precipitation) was subjected to
45% ammonium sulfate precipitation. The sample was centrifuged at 12,000 × g for 30 minutes,
and the 45% pellet was then re-suspended in 50 mM Tris-HCl (pH 7.0). The sample was then
dialyzed overnight to remove any excess ammonium sulfate. The desalted sample was then
loaded onto a Diethylaminoethyl(DEAE)-Cellulose (5 cm X 30 cm Anion Exchange, Sigma
Aldrich, D3764) column which had been equilibrated with 50mM Tris-HCl (pH 7.0). The protein
was eluted with an increasing NaCl gradient (0 – 500 mM NaCl in 50 mM Tris-HCl, pH 7.0).
Active fractions were confirmed via Sodium Dodecyl Sulfate – Poly-Acrylamide Gel
Electrophoresis (SDS-PAGE). They were consequently pooled, concentrated to approximate 3
mL and loaded onto a 1,6-diaminohexane (EAH) Sepharose Affinity (GE Healthcare Life
Sciences, 17-0569-03) column saturated with the F420 Cofactor. This was followed by a washing
with 300 mL of 20 mM Tris-HCl with 0.5 mM CHAPS, the protein was then eluted with an
increasing gradient from 20 mM Tris-HCl with 0.5 mM CHAPS to 600 mM NaCl – 20 mM Tris-
HCl with 0.5 mM CHAPS.
Active fractions were confirmed via SDS-PAGE. They were consequently pooled,
concentrated and loaded onto a High Sulfite (SO32-
) Cation Exchange column which had been
equilibrated with 50 mM HEPES buffer (pH 7.0). The protein was eluted with a linear gradient of
0 mM NaCl-50 mM HEPES to 600 mM NaCl-50 mM HEPES (pH 7.0).
The purified enzyme was identified with SDS-PAGE and activity assays, isolated and
stored at -80°C until it was ready for use. The enzyme concentration was measured using a
Bradford Protein Assay (Bio-Rad) by observing the wavelength shift of the Coomasie Brilliant
Blue G-250 from 465 nm to 595 nm when protein binding occurs.
22
Figure 2-5. Schematic of protein purification procedures
2.1.3 Site-directed mutagenesis of active site residues in FGD:
Several variants of the FGD enzyme have been successfully created in order to probe
their functionality. Using Quikchange Site-Directed Mutagenesis (Stratagene, Santa Clara, CA),
three single point mutations within FGD (H40A, W44F and W44A) were created (Figure 2-6).
However, this project focuses on the H40A mutant. As previously mentioned, based upon
crystallographic data, it is believed that Histidine 40 acts as an active site base, abstracting a
proton from the substrate, glucose-6-phosphate, facilitating the hydride transfer from the
substrate to the F420 Cofactor (Figure 2-4) 24
. The proposed mechanism of FGD also postulates
23
that following the initial proton abstraction from the O1 of G-6-P, the negatively charged reaction
intermediate which would be formed would be stabilized by interacting with Tryptophan 44
(W44) and possibly Glutamic Acid 13 (Glu13) depending on its protonation state. In order to
gain further insight into the hydride transfer mechanism of the FGD enzyme, aromatic and non-
aromatic amino acid substitutions were designed and synthesized (Figure 2-6). The W44
residue was converted to a phenylalanine (W44F) in order to investigate the role of the amino
acid in the catalyzed reaction of the FGD enzyme. The phenylalanine substitute only partially
reduces the aromaticity at the 44 position. However, the conversion of the W44 residue to an
Alanine (W44A) completely removes all aromaticity as the side chain is replaced with a neutral
Alanine residue. Studies with these mutations will perhaps grant further understanding of the
functionality of these residues as well as determining whether the aromaticity has an prominent
role in the stability of the intermediate. While the author was instrumental in the creation of
these variants, these studies are still on-going and beyond the scope of this thesis.
24
Figure 2-6. Sequence chromatograms showing successful mutation and conversion of designated amino acid residues in the active site of FGD
2.1.4 Crystallography:
G-6-P was added to H40A (10 mg/mL) to a final concentration of 1 mM G-6-P (10 times
the 100 μM Km24
). The search for the initial optimal crystallization conditions was undertaken
using forty-eight crystallization conditions using a PEG/Ion Screen (Hampton Research Inc.,
Aliso Viejo, CA) via the hanging-drop (vapor diffusion) method. The crystals grew best in five of
the forty eight conditions (Figure 2-7) shown below. Each drop consisted of 2 μL of the
enzyme/substrate sample (10 mg/mL) and 2 μL of the reservoir solution. However, these
crystals still have to be analyzed to determine if they are protein crystals.
25
Figure 2-7. Crystals formed from varying crystallization conditions of the H40A mutant variant. A - 0.2M Sodium fluoride, 20% w/v Polyethylene glycol 3,350; B - 0.2M Ammonium acetate, 20%
w/v Polyethylene glycol 3,350, C - 0.2M Sodium tartrate dibasic dihydrate, 20% w/v Polyethylene glycol 3,350; D - 0.2M Ammonium tartrate dibasic, 20% w/v Polyethylene glycol 3,350; E - 0.2M Potassium sodium tartrate tetrahydrate, 20% w/v Polyethylene glycol 3,350
26
CHAPTER 3
Results
3.1 Purification of wtFGD and mutant variants:
FGD was purified from E. coli as a recombinant system using ammonium sulfate
fractionation and several chromatographic methods. This method was effective in the
purification of the H40A mutant which is the focus of this work.
Figure 3-1. SDS-PAGE showing purification steps and a highlighted purified wtFGD. M – Marker; 1 – Streptomycin sulfate precipitation; 2 – Ammonium sulfate precipitation; 3 – DE-52
(Anion exchange chromatography); 4 – High S (Cation Exchange Chromatography)
The ammonium sulfate precipitation, although usually very harsh on protein, deemed to
be very effective in the isolation of FGD via salting out. Earlier studies (data not shown) showed
that the 45% ammonium sulfate precipitation (Lane 2) salted out all of the FGD protein while
leaving many others behind. However, the ammonium sulfate has to be removed immediately
after this step in order to maximize enzymatic activity.
The DEAE-Cellulose (DE-52) column was also very effective in increasing separations
between electrophoretic bands as observed via SDS-PAGE (Lane 3), while leaving the over-
expressed protein intact. Because FGD was purified from a recombinant system, the enzyme
was purified apo (without the cofactor). Therefore, the F420 Cofactor was purified from
27
Mycobacterium smegmatis and intercalated back into the protein. The F420-EAH Sepharose
column was vital as it was a means of merging apo-FGD with the cofactor. This step yielded a
highly purified enzyme as the affinity between FGD and the F420 Cofactor is very high (Figure 3-
1).
3.2 Active site mutations:
The design and development of mutant forms of FGD were successful as confirmed with
sequencing analysis (Sequetech DNA Sequencing Service, Mountain View, CA). The same
purification protocol that was used for wtFGD has been successful in purifying the H40A FGD
variant (Figure 3-2).
Figure 3-2. SDS-PAGE showing successful purification of wtFGD and H40A mutant variant.
Other lanes remain unchanged: Marker; 1 – Streptomycin sulfate precipitation; 2 – Ammonium sulfate precipitation; 3 – DE-52 (Anion exchange chromatography); Lane 4,5 and 6 – High S
(Cation Exchange Chromatography)
3.3 Steady-State Kinetics and pH Profile:
Following successful purification of wtFGD and the H40A FGD variant, steady-state
kinetics experiments were conducted to analyze the effect of the mutation on the catalytic
parameters of the enzyme. FGD activity was assayed by observing the decrease in the
absorbance of the oxidized F420 Cofactor at 420 nm upon reduction. The reagent volume and
28
concentrations for the assay is summarized in Table 3-1. The reaction kinetics were observed to
follow a Michaelis-Menten model (Table 3-2, Figure 3-3).
Table 3-1. Summary of concentration and volume parameters of kinetic assays
Reagent
Stock Concentration
(μM)
Final Concentration
(μM) Volume (μL)
Holo-FGD 11.4 0.05 8.7
G-6-P 1.0 x 104 Varied 800
K3PO4 50 mM N/A 1190
Table 3-2. Summary of kinetic parameters and comparison of wtFGD and H40A mutant variant
Parameter wtFGD H40A
kcat (1/s) 0.77 ± 0.04 0.89 ± 0.09
Km (μM) 53.8 ± 12.9 11 ± 4
Catalytic Efficiency (1/M·s) 1.43 x 104 6.6 x10
5
29
Figure 3-3. Steady-state kinetic plot and comparison of the wtFGD (Blue) and H40A (Red) variant
30
3.4 Conclusion
This is the first study of a substitution of the conserved H40 residue in FGD in which
kinetic properties are reported. The wtFGD had a kcat of 0.77 ± 0.04 s-1
while H40A FGD
exhibited a turnover number of 0.89 ± 0.09s-1
(Table 3-2). Therefore, within error, these values
are similar. However, the Km was observed to decrease significantly for the H40A FGD mutant
(wtFGD Km 53 ± 12; H40A Km 11 ± 4). This means that the H40A variant has a higher affinity for
the substrate as a smaller Km (assuming Km = KD) indicates a tighter binding between the
0
50
100
150
200
250
300
350
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
kcat (1
/s)
pH
H40A
wtFGD
Figure 3-4. pH profile of wtFGD and H40A mutant variant in 50 mM K3PO4 at 25°C
31
enzyme and substrate. Finally, the H40A variant was observed to exhibit a 45% increase in
catalytic efficiency (Table 3-2). It is possible that the mutation causes a rearrangement in the
enzyme’s conformation resulting in an increase of the mutant’s affinity for the substrate,
consequently increasing the efficiency of the enzyme. The H40A mutant was seen to be as
catalytically active as wtFGD, this was the first indication that this mutant may not serve as the
active site base for the FGD enzyme.
The pH profile of wtFGD, when juxtaposed with the H40A mutant, shows that the H40A
mutant exhibits very interesting dynamics. The wtFGD was observed to have pH optima (pH 4.5
and 8.0) while the H40A mutant was observed to have three (pH 4.5, 5.5 and 7.5). This
dissimilarity provides further insight into the catalytic behavior of the enzyme and possibly the
roles of other amino acids in close proximity to the substrate and cofactor. In general, the H40A
mutant was observed to perform optimally at lower a pH, while the wild type enzyme was
observed to perform optimally at a higher pH. It is plausible that the substitution of a neutral
Histidine at the 40th residue might affect the pKa of the acid/base catalyst in the active site
(E109) indirectly, resulting in a shift of the optimal pH. This information, along with the steady-
state data suggests that H40 is not serving as the active site base within FGD. However, while
the conserved H40 may not serve as an active site base, it is very likely that it serves other
purposes such as assisting in anchoring the substrate or possibly stabilizing an active site
intermediate, a function frequently identified with Tryptophan. This prompted further
investigation into the active site of FGD in order to locate other amino acid residues in close
proximity to the substrate which may serve as a general base to the enzyme.
32
Figure 3-5. Distance of the histidine amino acid residues in the active site from the modeled substrate, citrate in the active site of the FGD enzyme
24
Catalysis depends critically on the relationship between substrate and cofactor binding,
distance and spatial orientation. Upon further investigation into the active site of FGD, the H40
residue is seen to be further away from the substrate (modeled inhibitor, citrate) with a distance
of 4.59 Å as opposed to another Histidine with closer proximity, H260, which has a distance of
3.01 Å from the substrate (Figure 3-5). H260 is a conserved amino acid residue in the active
site of FGD. A multiple sequence alignment24
of 8 selected F420 Cofactor dependent enzymes
revealed the presence of a Histidine at the 260 position in 4 of the enzymes. Further studies on
H260 will hopefully identify the active site base.
33
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38
Biographical Information
Born and raised in Lagos, Nigeria, Tijani Osumah moved to Texas, USA on the 8th of
January, 2009 at the age of 16 to start his freshman year at the University of Texas at Arlington.
Since 2010, Tijani has worked under the supervision of Dr. Kayunta Johnson-Winters in
the Department of Chemistry and Biochemistry where he has researched on the recombinant
expression, purification and kinetic characterization of F420-dependent Glucose-6-phosphate
Dehydrogenase (FGD) enzyme from M. tuberculosis.
Tijani has received many awards throughout his career at the University of Texas at
Arlington including a University Scholar Award, John T. Murchison Award for Outstanding
Junior/ Pre-Health Professional student in the Department of Chemistry and Biochemistry and
the Igor Fraiberg Endowed Scholarship in Engineering.
Tijani will begin medical school with the September 2013 class at Ross University
School of Medicine, Portsmouth, Dominica.