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Identification of Protein-Protein Interactions of Glycine Oxidase (ThiO)

                   Brandon Turner, Lauren Pioppo

                         December 14, 2011
Abstract:

       Glycine Oxidase, or ThiO, from Bacillus subtilis catalyzes the FAD dependent oxidation of
glycine to imino-glycine in the thiamin biosynthesis pathway. In this pathway, the mechanism in
which iminoglycine and ThiS reaches the active site of ThiG remains unknown (Settembre et al
2003). Elucidation of this step in the pathway can aid in the development of drugs that target
thiamin biosynthesis in several pathogenic prokaryotes, such as P falciparum, the increasingly
resistant protozoan parasite that causes malaria. Here, we use recombinant His-tagged ORF811
from Bacillus muhlenbergius, a close homolog of B. subtilis ThiO, to analyze any protein:protein
interactions among ThiO and other enzymes involved in the passage of iminoglycine to ThiG
during thiamin biosynthesis. A pull-down assay of ORF811 and CFCE of B. muhlenbergius did not
reveal any stable protein:protein interactions with ORF811. Additionally, cross-linking studies
were performed to elucidate any transient interactions; a Western blot of these reactions
indicated possible dimer formation of ORF811, but did not reveal any relevant transient
interactions.
Introduction:

        Many molecular processes throughout the body, in all tissues, involve the interaction of
multiple proteins. These interactions can range from modification of other proteins, such as
phosphorylation or acylation, to the coupling of mechanistic steps by the close physical contact
of enzymes in a metabolic pathway. The latter can be accomplished by several methods
including the formation of large multi-enzyme complexes, such as pyruvate Dehydrogenase
(McMurry, Begley 2005), or unstable transient protein interactions (Krishnamurthy et al 2011).
Identifying protein-protein interactions has become a new practice of proteomics (Blagoev et al
2003) and can serve to elucidate how complex reactions involving reactive intermediates can
progress at a cellular level.

        Recently, a new organism named Bacillus muhlenbergius has been identified as a rare
soil bacterium inhabiting areas in Pennsylvania near Muhlenberg College, Allentown. This
organism is a close relative via genomic sequence of the more ubiquitous B. subtilis. A recent
project has been undertaken by this lab to identify the genomic characteristics of B.
muhlenbergius by elucidating the characteristics of several prokaryotic enzymes in hopes of
identifying enzymes that may be useful for biotechnological advances; one such enzyme is
Orf811. Bioinformatic analysis has identified Orf811 as a close homolog of Glycine Oxidase
(GO)1 from B. subtilis. GO is commonly referenced as ThiO for its oxidative role thiamin




biosynthesis. This enzyme is a homotetrameric protein that catalyzes the FAD dependent
oxidation of glycine to iminoglycine before being shuttled down the anabolic pathway
(Settembre et al 2003). However, several key factors in this progression of the pathway remain

1                       3
 GO: Glycine Oxidase. BS : Bissulfosuccinimidyl suberate. EDC: 1-ethyl-3-[dimethylaminopropyl]-carbodiimide.
CFCE: Cell-Free Crude Extract. BC: Bait Control. RC: Resin Control
unsolved. The imine product of GO is highly reactive in solvent and is readily hydrolyzed (Mortl
2006). This hydrolyzed product is not capable of being used in the pathway as the following
reaction of ThiS requires the imine product to continue. The method by which iminoglycine and
ThiS reach the active site of ThiG remains to be solved.

       The solved crystal structure of GO has shown that the enzyme contains a large channel
leading to the active site, which is buried within the hydrophobic core. However, the active site
is solvent exposed, indicating that the imine product cannot leave the active site without being
hydrolyzed (Mörtl et al 2004). Several enzymes that produce reactive intermediates have been
shown to contain structural characteristics that allow transfer of the species to the next active
site without exposure to other cellular components or solvent, such as the indole intermediate
in tryptophan synthase (Raushel et al 2003). This multifunction enzyme performs both portions
of the reaction with a protein ‘tunnel’ throughout the center to shuttle the reactive indole
molecule to the second active site. Other enzymes that produce reactive species have been
found to be part of large, multi-enzyme complexes, allowing the close proximity of the enzymes
in solvent to facilitate the transfer of the reactive species from one active site to another
(McMurry, Begley 2005). Given the reactive nature of iminoglycine and other protein
interactions within the thiamin biosynthesis pathway, such as that between ThiG and ThiS
(Settembre et al 2004), it is likely that some form of intermediate shuttling is occurring in order
to prevent hydrolysis prior to reacting with ThiS.

       In addition, the thiamin biosynthesis pathway has long been known as an antibiotic
target for several pathogenic prokaryotes that are unable to salvage vitamin B1, the most
notable being P. falciparum, the infections agent responsible for Malaria (Muller et al 2010).
Malaria was responsible for roughly 781,000 deaths in 2009 alone and is resistant to other
forms of antibiotic treatment as it is an obligate intracellular parasite (WHO 2011). Identifying
possible protein-protein interactions within the thiamin biosynthesis pathway can provide a
novel drug target for antibiotic research against this increasingly resistant pathogen.

       In this experiment, we attempted to elucidate the details of this passage of imino-
glycine to ThiS by performing a pull-down assay using His-tagged Orf811 and cell free crude
extracts from B. muhlenbergius to identify any stable protein-protein interactions between
other enzymes in the thiamin synthesis pathway. SDS-PAGE analysis revealed no visible
difference from control samples, indicating that no stable interactions had formed. In an
attempt to find any transient interactions between proteins, molecular crosslinkers were used
to identify putative protein-protein interactions involving ThiO. Western blot analysis of
samples crosslinked by BS3 showed possible dimer formation of ThiO, while EDC crosslinking
showed no bands. This study is important in identifying interactions between ThiO and other
enzymes in the thiamin synthesis pathway which can serve to explain how this reaction
progresses beyond the oxidation of glycine without loss of product to hydrolysis. Comparison of
this process in B. muhlenbergius to other prokaryotic organisms can serve to identify
evolutionary characteristics of this new organism and provide drug targets for P. falciparum.
Materials and Methods

Chemicals and Equipment

   All reaction kits were purchased from Thermo Scientific. Chemicals were purchased from
Qiagen, except for imidazole and Triton X-100 (ACROS), TRIS and 10% Tween 20 (BioRad), and
GelCode Stain and Coomassie Plus Protein Assay Reagent (Thermo Scientific). All high speed
(>10,000g) centrifugation was performed in the Sorvall RC6 High Speed Centrifuge, while slower
speeds were performed in the Eppendorf 5430. Cell Lysis was performed using a BeadBeater
(BioSpec).

Bioinformatics Analysis

       A Bioinformatic analysis of Orf811 was performed using ExPASy to obtain a partial
protein sequence from cDNA. The Basic Alignment Search Tool (BLASTP) was used to identify
potential homologs of Orf811 using the translated sequence. GenBank was then used to
identify the genomic context of GO.

Preparation of Bacillus muhlenbergius Cells

       Two cultures of B. muhlenbergius cells were grown in 1 liter of minimal media lacking
thiamin (5x M9 salts, Difco casamino acids, 20% glucose) at 30 C for about 48 hours. Cells were
harvested by centrifugation at 10,000xg for 30 min. Cell pellets were lysed by BeadBeating on
ice (12 cycles of beating for 15 seconds, followed by 45 seconds of rest) and centrifuged at
15,000xg to obtain CFCE. The concentration of the crude extract was determined using a
Bradford assay. Bovine serum albumin (BSA) was used for calibration.

Pull-Down Assay:

       A pull-down assay of B. muhlenbergius ORF811 was performed using the ProFound
Pull-Down PolyHis Protein:Protein Interaction Kit. Poly-His tagged ORF811 (3.5 mg/ml), a
generous gift from Dr. Keri Colabroy, Muhlenberg College, was used as bait protein, and
prepared cell-free crude extracts of B. muhlenbergius were used as a source of prey proteins.
Orf811 and CFCE were combined prior to use in the pull-down to a final protein concentration
of 2.35 mg/ml. This combined protein solution was also used for both cross-linking studies. The
pull-down was performed according to the protocol provided by Thermo Scientific. Briefly,
three 1.0 mL columns were prepared [(Bait Control (+Orf811, -CFCE), Resin Control (-Orf811,
+CFCE), Sample (+Orf811, -CFCE)] and treated with the corresponding proteins. Columns were
incubated at 4°C for 1 hour following the addition of Orf811 and an additional hour after
washing the column using the provided wash solution and treatment with CFCE. All flow
through was collected for analysis. Bound Orf811 was eluted from the columns using Imidazole
Elution Buffer (From kit).

Cross-Linking Studies

         Cell-free crude extracts of B. muhlenbergius and purified poly-His tagged ORF811 were
buffer exchanged into 20 mM potassium phosphate monobasic buffer (pH 7.8) using an Econo-
Pac 10 DG gel filtration column. This mixture was cross-linked with bissulfosuccinimidyl
suberate (BS3) following the protocol provided by Thermo Scientific. Cell-free crude extracts of
B. muhlenbergius and purified poly-His tagged ORF811 were buffer exchanged into 0.1 M MES
buffer (2-[N-morpholino]ethane sulfonic acid, pH 4.94) using an Econo-Pac 10 DG gel filtration
column. This mixture was cross-linked with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride (EDC) following the protocol provided by Thermo Scientific.

         Briefly, 10mg EDC was added to 1 mL of ultra-pure (miliQ) H2O to prepare the
crosslinking solution. 100 uL of EDC solution was added to a 1 mL sample of combined protein
solution and incubated at room temperature for two hours. The reaction was desalted by use of
Amicron 4.0 mL Ultra Filtration with addition of MES buffer, centrifuged 3x at 7100xg for 10
min.

         For BS3, a 12.5 mM solution was prepared in a 50-molar excess to protein concentration
by adding 277 uL of 20 mM Potassium Phosphate buffer to 2 mg of BS 3. 100 uL of BS3 solution
was added to 500 uL of combined protein sample and incubated at room temperature for 30
min. Tris-HCl buffer was added to quench the reaction and the quenched reaction was stored at
-20°C.
SDS-PAGE and Western Blot Analysis

       Pull-down and crosslinking samples were analyzed by SDS-PAGE Gel Electrophoresis.
Samples were run on two 4-20% polyacrylamide gels. The gel containing pull-down samples was
stained using GelCode stain while the gel containing the crosslinking samples was
electroblotted to a membrane. The membrane was subjected to staining with Anti-His HRP
Conjugate antibodies.
Results:

Bioinformatics Analysis of Orf811

       In order to glance at Orf811’s possible structure and function, a bioinformatics approach
was used to find homologs. The fragment of sequenced genome was translated using ExPASy
and run through BLASTP which showed a high relation to Glycine Oxidase from Bacillus subtilis
(Max Score: 447, E-value: 2e-156, top hit). This homolog is ThiO, an FAD dependent oxidase,
and is implicated in thiamine biosynthesis by its close proximity to ThiS, a sulfur carrier, and
ThiF, which is involved in thiamine/molybdopterin sytnthesis. Structural data shows it exists as
a homotetramer (3). Based on its sequence similarity, it is likely that Orf811 will be similar in
function to ThiO.

Preparation and Purification of Bacillus muhlenbergius Cell Lysate

       Cultures of B. muhlenbergius were harvested at OD600 = 0.6 and lysed via BeadBeating to
obtain CFCE. Cellular protein was purified to 12.46 mg/mL and stored with glycerol at -20°C.
Protein samples were aliquoted in 800uL amounts for future use. Although the presence of
proteins involved in thiamine biosynthesis was unable to be determined, dark bands close to
the molecular weights of involved proteins (ThiS: 7.89 kDa, ThiG, 26.9 kDa) suggest an increase
in expression of these enzymes.

Pull Down Assay

       A pull-down assay involving His-tagged Orf811 was performed to locate stable
protein:protein interactions within the thiamine biosynthetic pathway. SDS-PAGE analysis of all
samples, including flow through and positive controls was performed to view any bound
proteins. Control samples consisted of Bait Control (BC) with no prey protein solution added to
the column and Resin Control (RC) with no Orf811 bound to the column. Flow thru after the
addition of both bait and prey proteins were collected for analysis. Gel bands for experimental
samples were identical to that of bait control (fig. 1), indicating that no stable interactions were
present. The double band shown for Orf811 is consistent with an addition of a small protein;
however, with no difference between sample and control lanes, no additional proteins seem to
have been purified. Due to lack of binding, gel bands were not excised for peptide mass
fingerprinting.
Cross-Linking Studies Cross-linking studies involving a protein mix of Orf811 and CFCE
were performed using BS3 and EDC. In order to perform these studies, protein solutions were
buffer exchanged into KPO3 monobasic buffer to avoid cross-linker interactions with solvent.
The cross-linked solutions were analyzed via western blotting using His-tag specific antibodies
to observe any changes in molecular weight of Orf811. No bands were observed for EDC cross-
linking, but two additional bands were present for BS3 (fig. 2). Both bands are higher in
molecular weight than Orf811 but do not correspond to an addition of 7.2 kDa (ThiS) or 26.9
kDa (ThiG) (Settembre et al 2004). Molecular weights are consistent with dimer and trimer
formation of GO due to the even spacing of gel bands and their apparent increase in molecular
weight of 47 kDa.
Discussion:

       We present here an attempt to identify stable and transient protein:protein interactions
between the homotetrameric protein, ThiO and other enzymes in the thiamin biosynthesis
pathway. Although ThiO is known to catalyze the FAD dependent oxidation of glycine to imino-
glycine in this pathway (Settembre et al 2003), several components of the progression remain
unsolved. One unsolved component of this pathway includes the highly reactive imine product
of ThiO; although this product is readily hydrolyzed, the next reaction in the pathway requires
the non-hydrolyzed imine to occur. This reaction, which includes the binding of both
iminoglycine and ThiS to the active site of ThiG, poses the question as to how ThiS and
iminoglycine reach ThiG (Mörtl et al 2004). Since the active site of ThiO is known to be solvent
exposed (Mörtl et al 2004), it is possible that the imine product is transferred to the active site
of ThiG through intermediate shuffling to prevent hydrolysis of iminoglycine (Settembre et al
2004). Analysis of the protein:protein interactions between ThiO and other enzymes in the
thiamin biosynthesis pathway, such as ThiS, may aid in elucidating this potential intermediate
shuffling and the further mapping of the pathway mechanisms. This work includes a pull-down
assay and cross-linking studies to study both stable and transient protein:protein interactions,
respectively.

       The newly discovered species of bacteria, Bacillus muhlenbergius, was used in this study.
Prior characterization of this species has revealed a novel enzyme, ORF811, which was used in
all experiments. By utilizing the Basic Local Alignment Search Tool, we found that closely
related homologs of ORF811 were annotated as glycine oxidases, indicating that ORF811 may
also function as a glycine oxidase. Using the BRENDA enzyme database, it was found that a
glycine oxidation reaction catalyzed by an ORF811 homolog consists of glycine, water, and
molecular oxygen reacting to form glycoxylate, ammonia, and hydrogen peroxide; this provides
some insight as to what type of reaction ORF811 may catalyze. Additionally, a genomic context
analysis of closely related homologs of ORF811 revealed that the homologous gene ThiO, as
well as its surrounding genes, appears to function in thiamine biosynthesis; this indicates that
ORF811 may play similar roles in metabolism. Thus, the apparent close homology between
ORF811 and ThiO allowed us to use recombinant His-tagged ORF811 to facilitate the study of
protein:protein interactions between ThiO and other enzymes in the thiamin biosynthesis
pathway.

       The study was initiated by growing B. muhlenbergius in nutrient deficient media. This
forced the bacteria to produce large amounts of their own thiamin, as well as the proteins
implicated in thiamin biosynthesis, ideally to improve binding of ORF811 to its potential
partners in the pathway. Growth and harvesting of B. muhlenbergius was successful, and 9.5 ml
of cell lysate was recovered from 2 L of culture; a Bradford assay revealed the concentration of
this CFCE to be 12.46 mg/ml.

       To study any stable protein:protein interactions, a pull-down assay was conducted,
followed by an SDS-polyacrylamide gel to analyze results. By immobilizing the recombinant His-
tagged ORF811 bait to a column with a cobalt chelate resin, we were able to run a sample of B.
muhlenbergius cell-free crude extract through the column to determine if any proteins in the
extract bound to the immobilized ORF811. However, an SDS-PAGE gel of the eluant revealed no
difference between the bait control, which contained only ORF811 bound to the resin, and the
experimental sample, which contained both the ORF811 bait and the CFCE prey (Fig. 1). Thus,
no stable protein-protein interactions between this close homolog of ThiO and other enzymes
in the thiamin biosynthesis pathway were observed.

       Cross-linking studies allowed any transient protein:protein interactions with ORF811 to
be assessed. Following the cross-linking reaction, a Western blot was performed to compare
the size of the band to a control sample of just ORF811; an increase in size would indicate that
binding may have occurred. A solution of ORF811 and B. muhlenbergius CFCE were cross-linked
with both BS3 and EDC. When EDC was used as the cross-linker, no signal was observed on the
Western blot. Prior to the cross-linking reaction, the solution of CFCE and ORF811 was buffer
exchanged three times, which resulted in a significant decrease in protein concentration. Thus,
it is possible that the concentration of protein was too low for any cross-linking to be visualized
on the blot. Another EDC cross-linking reaction with a higher concentration of protein is needed
to confirm this conclusion. Although bands were observed when BS 3 was used as the cross-
linker, it is not likely that these bands correspond to any relevant protein:protein interactions
with ThiO. Previous studies have indicated that ThiO is a 47 kDa protein (Job et. al. 2001), and
the three bands on the Western blot (Fig. 2) appear to be equally spaced apart by about 47
kDa; the bands are about 47 kDa, 94 kDa, and 141 kDa. This 47 kDa spacing between bands
suggests that ORF811 formed a homodimer, and is thus not indicative of any relevant transient
protein:protein interactions. However, this conclusion needs to be confirmed by protein
sequencing of the bands.

       Although no stable or transient protein:protein interactions with B. muhlenbergius
ORF811 and other enzymes involved in thiamin biosynthesis were observed, further research is
needed to investigate these possible interactions. Addition of a glycine or sulfite substrate may
facilitate the close relation of the possible binding partners, such as ThiS, and thus aid in
assessing these interactions. Further elucidation of the method in which iminoglycine and ThiS
are passed to the active site of ThiG may provide a potential antibiotic target for pathogens that
are susceptible to antibiotics that target thiamin synthesis, such as P. falciparum, the infections
agent responsible for Malaria (Muller et al 2010).



References:

   1. Job V, Marcone GL, Pilone MS, Pollegioni, L. 2001. . Glycine oxidase from Bacillus
               subtilis. Characterization of a new flavoprotein. Biological Chemistry [Internet].
               [Cited 2011 Oct 28] 277(9):6985-93. Available from:
               http://www.jbc.org/content/277/9/6985.long#cited-by
   2. Krishnamurthy M, Dugan A, Nowkoye A, Fung YH, Lancia JK, Majmudar CY and Mapp AK.
               Caught in the act: covalent crosslinking captures activator-coactivator
               interactions in vivo (2011). ACS Chem Biol. [Epub ahead of print].
   3. McMurry, John and Begley, Tadhg. The Organic Chemistry of Biological Pathways.
               Englewood, Colorado: Roberts and Company Publishers. 2005.
4. Mortl M, Diderichs K, Welte W, Molla G, Motteran L, Andriolo G, Pilone MS and
           Pollegioni L. Structure-Function Correlation in Glycine Oxidase from Bacillus
           subtilis. J. Biol Chem. 2004, 28(279), 29718-29727.
5. Mortl, Mario. Substrate specificity of Glycine Oxidase and protein interaction specificity
           of the neuronal cell adhesion molecule TAG-1. University of Konstanz. 2006.
           Dissertation.
6. Muller IB, Hyde JE, Wrenger C. Vitamin B Metabolism in Plasmodium falciparum as a
           source of drug targets (2010). Trends in Parasitology. 1(26), 35-43
7. Raushel FM, Thoden JB, and Holden HM. Enzymes with Molecular Tunnels (2003). Acc.
           Chem. Res. 36, 539 - 548
8. Setembre EC, Dorrestein PC, Zhai H, Chatterjee A, McLafferty FW, Begley TP and Ealick
           SE. Thiamin Biosynthesis in Bacillus subtilis: Structure of the Thiazole
           Synthase/Sulfur Carrier Protein Complex (2004). Biochemistry. 43, 11647 –
           11657.
9. Settembre EC, Dorrestein PC, Park JH, Augustine AM, Begley TP and Ealick SE. Structural
           and Mechanistic Studies on ThiO, a Glycine Oxidase Essential for Thiamin
           Biosynthesis in Bacillus subtilis (2003). Biochemistry. 42: 2971 – 81.
10. World Health Organization. Malaria fact sheet. Last updated Oct. 2011.
           http://www.who.int/mediacentre/factsheets/fs094/en/index.html
11. Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ and Mann M. A proteomics
           strategy to elucidate functional protein-protein interactions applied to EGF
           signaling (2003). Nat Biotechnol. 21(3) 315-318.
12. Settembre EC, Dorrestein PC, Zhai H, Chatterjee A, McLafferty FW, Begley TP, and Ealick
           SE. Thiamin Biosynthesis in Bacillus subtilis: Structure of the Thiazole
           Synthase/Sulfur Carrier Protein Complex (2004). Biochemistry. 43: 11647-11657

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Protein Protein Interactions Of Glycine Oxidase (Thi O)

  • 1. Identification of Protein-Protein Interactions of Glycine Oxidase (ThiO) Brandon Turner, Lauren Pioppo December 14, 2011
  • 2. Abstract: Glycine Oxidase, or ThiO, from Bacillus subtilis catalyzes the FAD dependent oxidation of glycine to imino-glycine in the thiamin biosynthesis pathway. In this pathway, the mechanism in which iminoglycine and ThiS reaches the active site of ThiG remains unknown (Settembre et al 2003). Elucidation of this step in the pathway can aid in the development of drugs that target thiamin biosynthesis in several pathogenic prokaryotes, such as P falciparum, the increasingly resistant protozoan parasite that causes malaria. Here, we use recombinant His-tagged ORF811 from Bacillus muhlenbergius, a close homolog of B. subtilis ThiO, to analyze any protein:protein interactions among ThiO and other enzymes involved in the passage of iminoglycine to ThiG during thiamin biosynthesis. A pull-down assay of ORF811 and CFCE of B. muhlenbergius did not reveal any stable protein:protein interactions with ORF811. Additionally, cross-linking studies were performed to elucidate any transient interactions; a Western blot of these reactions indicated possible dimer formation of ORF811, but did not reveal any relevant transient interactions.
  • 3. Introduction: Many molecular processes throughout the body, in all tissues, involve the interaction of multiple proteins. These interactions can range from modification of other proteins, such as phosphorylation or acylation, to the coupling of mechanistic steps by the close physical contact of enzymes in a metabolic pathway. The latter can be accomplished by several methods including the formation of large multi-enzyme complexes, such as pyruvate Dehydrogenase (McMurry, Begley 2005), or unstable transient protein interactions (Krishnamurthy et al 2011). Identifying protein-protein interactions has become a new practice of proteomics (Blagoev et al 2003) and can serve to elucidate how complex reactions involving reactive intermediates can progress at a cellular level. Recently, a new organism named Bacillus muhlenbergius has been identified as a rare soil bacterium inhabiting areas in Pennsylvania near Muhlenberg College, Allentown. This organism is a close relative via genomic sequence of the more ubiquitous B. subtilis. A recent project has been undertaken by this lab to identify the genomic characteristics of B. muhlenbergius by elucidating the characteristics of several prokaryotic enzymes in hopes of identifying enzymes that may be useful for biotechnological advances; one such enzyme is Orf811. Bioinformatic analysis has identified Orf811 as a close homolog of Glycine Oxidase (GO)1 from B. subtilis. GO is commonly referenced as ThiO for its oxidative role thiamin biosynthesis. This enzyme is a homotetrameric protein that catalyzes the FAD dependent oxidation of glycine to iminoglycine before being shuttled down the anabolic pathway (Settembre et al 2003). However, several key factors in this progression of the pathway remain 1 3 GO: Glycine Oxidase. BS : Bissulfosuccinimidyl suberate. EDC: 1-ethyl-3-[dimethylaminopropyl]-carbodiimide. CFCE: Cell-Free Crude Extract. BC: Bait Control. RC: Resin Control
  • 4. unsolved. The imine product of GO is highly reactive in solvent and is readily hydrolyzed (Mortl 2006). This hydrolyzed product is not capable of being used in the pathway as the following reaction of ThiS requires the imine product to continue. The method by which iminoglycine and ThiS reach the active site of ThiG remains to be solved. The solved crystal structure of GO has shown that the enzyme contains a large channel leading to the active site, which is buried within the hydrophobic core. However, the active site is solvent exposed, indicating that the imine product cannot leave the active site without being hydrolyzed (Mörtl et al 2004). Several enzymes that produce reactive intermediates have been shown to contain structural characteristics that allow transfer of the species to the next active site without exposure to other cellular components or solvent, such as the indole intermediate in tryptophan synthase (Raushel et al 2003). This multifunction enzyme performs both portions of the reaction with a protein ‘tunnel’ throughout the center to shuttle the reactive indole molecule to the second active site. Other enzymes that produce reactive species have been found to be part of large, multi-enzyme complexes, allowing the close proximity of the enzymes in solvent to facilitate the transfer of the reactive species from one active site to another (McMurry, Begley 2005). Given the reactive nature of iminoglycine and other protein interactions within the thiamin biosynthesis pathway, such as that between ThiG and ThiS (Settembre et al 2004), it is likely that some form of intermediate shuttling is occurring in order to prevent hydrolysis prior to reacting with ThiS. In addition, the thiamin biosynthesis pathway has long been known as an antibiotic target for several pathogenic prokaryotes that are unable to salvage vitamin B1, the most notable being P. falciparum, the infections agent responsible for Malaria (Muller et al 2010). Malaria was responsible for roughly 781,000 deaths in 2009 alone and is resistant to other forms of antibiotic treatment as it is an obligate intracellular parasite (WHO 2011). Identifying possible protein-protein interactions within the thiamin biosynthesis pathway can provide a novel drug target for antibiotic research against this increasingly resistant pathogen. In this experiment, we attempted to elucidate the details of this passage of imino- glycine to ThiS by performing a pull-down assay using His-tagged Orf811 and cell free crude
  • 5. extracts from B. muhlenbergius to identify any stable protein-protein interactions between other enzymes in the thiamin synthesis pathway. SDS-PAGE analysis revealed no visible difference from control samples, indicating that no stable interactions had formed. In an attempt to find any transient interactions between proteins, molecular crosslinkers were used to identify putative protein-protein interactions involving ThiO. Western blot analysis of samples crosslinked by BS3 showed possible dimer formation of ThiO, while EDC crosslinking showed no bands. This study is important in identifying interactions between ThiO and other enzymes in the thiamin synthesis pathway which can serve to explain how this reaction progresses beyond the oxidation of glycine without loss of product to hydrolysis. Comparison of this process in B. muhlenbergius to other prokaryotic organisms can serve to identify evolutionary characteristics of this new organism and provide drug targets for P. falciparum.
  • 6. Materials and Methods Chemicals and Equipment All reaction kits were purchased from Thermo Scientific. Chemicals were purchased from Qiagen, except for imidazole and Triton X-100 (ACROS), TRIS and 10% Tween 20 (BioRad), and GelCode Stain and Coomassie Plus Protein Assay Reagent (Thermo Scientific). All high speed (>10,000g) centrifugation was performed in the Sorvall RC6 High Speed Centrifuge, while slower speeds were performed in the Eppendorf 5430. Cell Lysis was performed using a BeadBeater (BioSpec). Bioinformatics Analysis A Bioinformatic analysis of Orf811 was performed using ExPASy to obtain a partial protein sequence from cDNA. The Basic Alignment Search Tool (BLASTP) was used to identify potential homologs of Orf811 using the translated sequence. GenBank was then used to identify the genomic context of GO. Preparation of Bacillus muhlenbergius Cells Two cultures of B. muhlenbergius cells were grown in 1 liter of minimal media lacking thiamin (5x M9 salts, Difco casamino acids, 20% glucose) at 30 C for about 48 hours. Cells were harvested by centrifugation at 10,000xg for 30 min. Cell pellets were lysed by BeadBeating on ice (12 cycles of beating for 15 seconds, followed by 45 seconds of rest) and centrifuged at 15,000xg to obtain CFCE. The concentration of the crude extract was determined using a Bradford assay. Bovine serum albumin (BSA) was used for calibration. Pull-Down Assay: A pull-down assay of B. muhlenbergius ORF811 was performed using the ProFound Pull-Down PolyHis Protein:Protein Interaction Kit. Poly-His tagged ORF811 (3.5 mg/ml), a generous gift from Dr. Keri Colabroy, Muhlenberg College, was used as bait protein, and prepared cell-free crude extracts of B. muhlenbergius were used as a source of prey proteins.
  • 7. Orf811 and CFCE were combined prior to use in the pull-down to a final protein concentration of 2.35 mg/ml. This combined protein solution was also used for both cross-linking studies. The pull-down was performed according to the protocol provided by Thermo Scientific. Briefly, three 1.0 mL columns were prepared [(Bait Control (+Orf811, -CFCE), Resin Control (-Orf811, +CFCE), Sample (+Orf811, -CFCE)] and treated with the corresponding proteins. Columns were incubated at 4°C for 1 hour following the addition of Orf811 and an additional hour after washing the column using the provided wash solution and treatment with CFCE. All flow through was collected for analysis. Bound Orf811 was eluted from the columns using Imidazole Elution Buffer (From kit). Cross-Linking Studies Cell-free crude extracts of B. muhlenbergius and purified poly-His tagged ORF811 were buffer exchanged into 20 mM potassium phosphate monobasic buffer (pH 7.8) using an Econo- Pac 10 DG gel filtration column. This mixture was cross-linked with bissulfosuccinimidyl suberate (BS3) following the protocol provided by Thermo Scientific. Cell-free crude extracts of B. muhlenbergius and purified poly-His tagged ORF811 were buffer exchanged into 0.1 M MES buffer (2-[N-morpholino]ethane sulfonic acid, pH 4.94) using an Econo-Pac 10 DG gel filtration column. This mixture was cross-linked with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) following the protocol provided by Thermo Scientific. Briefly, 10mg EDC was added to 1 mL of ultra-pure (miliQ) H2O to prepare the crosslinking solution. 100 uL of EDC solution was added to a 1 mL sample of combined protein solution and incubated at room temperature for two hours. The reaction was desalted by use of Amicron 4.0 mL Ultra Filtration with addition of MES buffer, centrifuged 3x at 7100xg for 10 min. For BS3, a 12.5 mM solution was prepared in a 50-molar excess to protein concentration by adding 277 uL of 20 mM Potassium Phosphate buffer to 2 mg of BS 3. 100 uL of BS3 solution was added to 500 uL of combined protein sample and incubated at room temperature for 30 min. Tris-HCl buffer was added to quench the reaction and the quenched reaction was stored at -20°C.
  • 8. SDS-PAGE and Western Blot Analysis Pull-down and crosslinking samples were analyzed by SDS-PAGE Gel Electrophoresis. Samples were run on two 4-20% polyacrylamide gels. The gel containing pull-down samples was stained using GelCode stain while the gel containing the crosslinking samples was electroblotted to a membrane. The membrane was subjected to staining with Anti-His HRP Conjugate antibodies.
  • 9. Results: Bioinformatics Analysis of Orf811 In order to glance at Orf811’s possible structure and function, a bioinformatics approach was used to find homologs. The fragment of sequenced genome was translated using ExPASy and run through BLASTP which showed a high relation to Glycine Oxidase from Bacillus subtilis (Max Score: 447, E-value: 2e-156, top hit). This homolog is ThiO, an FAD dependent oxidase, and is implicated in thiamine biosynthesis by its close proximity to ThiS, a sulfur carrier, and ThiF, which is involved in thiamine/molybdopterin sytnthesis. Structural data shows it exists as a homotetramer (3). Based on its sequence similarity, it is likely that Orf811 will be similar in function to ThiO. Preparation and Purification of Bacillus muhlenbergius Cell Lysate Cultures of B. muhlenbergius were harvested at OD600 = 0.6 and lysed via BeadBeating to obtain CFCE. Cellular protein was purified to 12.46 mg/mL and stored with glycerol at -20°C. Protein samples were aliquoted in 800uL amounts for future use. Although the presence of proteins involved in thiamine biosynthesis was unable to be determined, dark bands close to the molecular weights of involved proteins (ThiS: 7.89 kDa, ThiG, 26.9 kDa) suggest an increase in expression of these enzymes. Pull Down Assay A pull-down assay involving His-tagged Orf811 was performed to locate stable protein:protein interactions within the thiamine biosynthetic pathway. SDS-PAGE analysis of all samples, including flow through and positive controls was performed to view any bound proteins. Control samples consisted of Bait Control (BC) with no prey protein solution added to the column and Resin Control (RC) with no Orf811 bound to the column. Flow thru after the addition of both bait and prey proteins were collected for analysis. Gel bands for experimental samples were identical to that of bait control (fig. 1), indicating that no stable interactions were present. The double band shown for Orf811 is consistent with an addition of a small protein; however, with no difference between sample and control lanes, no additional proteins seem to
  • 10. have been purified. Due to lack of binding, gel bands were not excised for peptide mass fingerprinting.
  • 11. Cross-Linking Studies Cross-linking studies involving a protein mix of Orf811 and CFCE were performed using BS3 and EDC. In order to perform these studies, protein solutions were buffer exchanged into KPO3 monobasic buffer to avoid cross-linker interactions with solvent. The cross-linked solutions were analyzed via western blotting using His-tag specific antibodies to observe any changes in molecular weight of Orf811. No bands were observed for EDC cross- linking, but two additional bands were present for BS3 (fig. 2). Both bands are higher in molecular weight than Orf811 but do not correspond to an addition of 7.2 kDa (ThiS) or 26.9 kDa (ThiG) (Settembre et al 2004). Molecular weights are consistent with dimer and trimer formation of GO due to the even spacing of gel bands and their apparent increase in molecular weight of 47 kDa.
  • 12. Discussion: We present here an attempt to identify stable and transient protein:protein interactions between the homotetrameric protein, ThiO and other enzymes in the thiamin biosynthesis pathway. Although ThiO is known to catalyze the FAD dependent oxidation of glycine to imino- glycine in this pathway (Settembre et al 2003), several components of the progression remain unsolved. One unsolved component of this pathway includes the highly reactive imine product of ThiO; although this product is readily hydrolyzed, the next reaction in the pathway requires the non-hydrolyzed imine to occur. This reaction, which includes the binding of both iminoglycine and ThiS to the active site of ThiG, poses the question as to how ThiS and iminoglycine reach ThiG (Mörtl et al 2004). Since the active site of ThiO is known to be solvent exposed (Mörtl et al 2004), it is possible that the imine product is transferred to the active site of ThiG through intermediate shuffling to prevent hydrolysis of iminoglycine (Settembre et al 2004). Analysis of the protein:protein interactions between ThiO and other enzymes in the thiamin biosynthesis pathway, such as ThiS, may aid in elucidating this potential intermediate shuffling and the further mapping of the pathway mechanisms. This work includes a pull-down assay and cross-linking studies to study both stable and transient protein:protein interactions, respectively. The newly discovered species of bacteria, Bacillus muhlenbergius, was used in this study. Prior characterization of this species has revealed a novel enzyme, ORF811, which was used in all experiments. By utilizing the Basic Local Alignment Search Tool, we found that closely related homologs of ORF811 were annotated as glycine oxidases, indicating that ORF811 may also function as a glycine oxidase. Using the BRENDA enzyme database, it was found that a glycine oxidation reaction catalyzed by an ORF811 homolog consists of glycine, water, and molecular oxygen reacting to form glycoxylate, ammonia, and hydrogen peroxide; this provides some insight as to what type of reaction ORF811 may catalyze. Additionally, a genomic context analysis of closely related homologs of ORF811 revealed that the homologous gene ThiO, as
  • 13. well as its surrounding genes, appears to function in thiamine biosynthesis; this indicates that ORF811 may play similar roles in metabolism. Thus, the apparent close homology between ORF811 and ThiO allowed us to use recombinant His-tagged ORF811 to facilitate the study of protein:protein interactions between ThiO and other enzymes in the thiamin biosynthesis pathway. The study was initiated by growing B. muhlenbergius in nutrient deficient media. This forced the bacteria to produce large amounts of their own thiamin, as well as the proteins implicated in thiamin biosynthesis, ideally to improve binding of ORF811 to its potential partners in the pathway. Growth and harvesting of B. muhlenbergius was successful, and 9.5 ml of cell lysate was recovered from 2 L of culture; a Bradford assay revealed the concentration of this CFCE to be 12.46 mg/ml. To study any stable protein:protein interactions, a pull-down assay was conducted, followed by an SDS-polyacrylamide gel to analyze results. By immobilizing the recombinant His- tagged ORF811 bait to a column with a cobalt chelate resin, we were able to run a sample of B. muhlenbergius cell-free crude extract through the column to determine if any proteins in the extract bound to the immobilized ORF811. However, an SDS-PAGE gel of the eluant revealed no difference between the bait control, which contained only ORF811 bound to the resin, and the experimental sample, which contained both the ORF811 bait and the CFCE prey (Fig. 1). Thus, no stable protein-protein interactions between this close homolog of ThiO and other enzymes in the thiamin biosynthesis pathway were observed. Cross-linking studies allowed any transient protein:protein interactions with ORF811 to be assessed. Following the cross-linking reaction, a Western blot was performed to compare the size of the band to a control sample of just ORF811; an increase in size would indicate that binding may have occurred. A solution of ORF811 and B. muhlenbergius CFCE were cross-linked with both BS3 and EDC. When EDC was used as the cross-linker, no signal was observed on the Western blot. Prior to the cross-linking reaction, the solution of CFCE and ORF811 was buffer exchanged three times, which resulted in a significant decrease in protein concentration. Thus, it is possible that the concentration of protein was too low for any cross-linking to be visualized
  • 14. on the blot. Another EDC cross-linking reaction with a higher concentration of protein is needed to confirm this conclusion. Although bands were observed when BS 3 was used as the cross- linker, it is not likely that these bands correspond to any relevant protein:protein interactions with ThiO. Previous studies have indicated that ThiO is a 47 kDa protein (Job et. al. 2001), and the three bands on the Western blot (Fig. 2) appear to be equally spaced apart by about 47 kDa; the bands are about 47 kDa, 94 kDa, and 141 kDa. This 47 kDa spacing between bands suggests that ORF811 formed a homodimer, and is thus not indicative of any relevant transient protein:protein interactions. However, this conclusion needs to be confirmed by protein sequencing of the bands. Although no stable or transient protein:protein interactions with B. muhlenbergius ORF811 and other enzymes involved in thiamin biosynthesis were observed, further research is needed to investigate these possible interactions. Addition of a glycine or sulfite substrate may facilitate the close relation of the possible binding partners, such as ThiS, and thus aid in assessing these interactions. Further elucidation of the method in which iminoglycine and ThiS are passed to the active site of ThiG may provide a potential antibiotic target for pathogens that are susceptible to antibiotics that target thiamin synthesis, such as P. falciparum, the infections agent responsible for Malaria (Muller et al 2010). References: 1. Job V, Marcone GL, Pilone MS, Pollegioni, L. 2001. . Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein. Biological Chemistry [Internet]. [Cited 2011 Oct 28] 277(9):6985-93. Available from: http://www.jbc.org/content/277/9/6985.long#cited-by 2. Krishnamurthy M, Dugan A, Nowkoye A, Fung YH, Lancia JK, Majmudar CY and Mapp AK. Caught in the act: covalent crosslinking captures activator-coactivator interactions in vivo (2011). ACS Chem Biol. [Epub ahead of print]. 3. McMurry, John and Begley, Tadhg. The Organic Chemistry of Biological Pathways. Englewood, Colorado: Roberts and Company Publishers. 2005.
  • 15. 4. Mortl M, Diderichs K, Welte W, Molla G, Motteran L, Andriolo G, Pilone MS and Pollegioni L. Structure-Function Correlation in Glycine Oxidase from Bacillus subtilis. J. Biol Chem. 2004, 28(279), 29718-29727. 5. Mortl, Mario. Substrate specificity of Glycine Oxidase and protein interaction specificity of the neuronal cell adhesion molecule TAG-1. University of Konstanz. 2006. Dissertation. 6. Muller IB, Hyde JE, Wrenger C. Vitamin B Metabolism in Plasmodium falciparum as a source of drug targets (2010). Trends in Parasitology. 1(26), 35-43 7. Raushel FM, Thoden JB, and Holden HM. Enzymes with Molecular Tunnels (2003). Acc. Chem. Res. 36, 539 - 548 8. Setembre EC, Dorrestein PC, Zhai H, Chatterjee A, McLafferty FW, Begley TP and Ealick SE. Thiamin Biosynthesis in Bacillus subtilis: Structure of the Thiazole Synthase/Sulfur Carrier Protein Complex (2004). Biochemistry. 43, 11647 – 11657. 9. Settembre EC, Dorrestein PC, Park JH, Augustine AM, Begley TP and Ealick SE. Structural and Mechanistic Studies on ThiO, a Glycine Oxidase Essential for Thiamin Biosynthesis in Bacillus subtilis (2003). Biochemistry. 42: 2971 – 81. 10. World Health Organization. Malaria fact sheet. Last updated Oct. 2011. http://www.who.int/mediacentre/factsheets/fs094/en/index.html 11. Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ and Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling (2003). Nat Biotechnol. 21(3) 315-318. 12. Settembre EC, Dorrestein PC, Zhai H, Chatterjee A, McLafferty FW, Begley TP, and Ealick SE. Thiamin Biosynthesis in Bacillus subtilis: Structure of the Thiazole Synthase/Sulfur Carrier Protein Complex (2004). Biochemistry. 43: 11647-11657