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 GO: Glycine Oxidase. BS

3: 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 BS3. 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 BS3 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).

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