 The document describes cloning, expressing, and purifying a glutathione S-transferase (GST) from Pseudomonas putida KT2440 and investigating its potential role in iron metal bioremediation. Key findings include: - The GST gene (PP_2933) was cloned, expressed producing a 31kDa protein, and purified using ion exchange chromatography. - The purified GST showed activity with certain substrates, including ethacrynic acid and cumene peroxide, indicating a role in oxidative stress response. - Cells expressing the recombinant GST showed increased resistance to iron, suggesting it may bind iron complexes and aid in bioremediation of iron toxicity.

1. MOLECULAR CLONING, EXPRESSION AND PURIFICATION OF A
GLUTATHIONE S-TRANSFERASE (GST) FROM PSEUDOMONAS PUTIDA KT2440 AND
ITS POTENTIAL APPLICATION IN IRON METAL BIOREMEDIATION
NASIRU ABDULLAHI, ZAZALI ALIAS and SALMAH ISMAIL
Faculty of Science, Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur
Methodology
Results and Discussion
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T2O CDNB DCNB FESO4 EA CUOOH EPNP PBO H24D DCM T2HEX
In silico identification of GST
genes Pseudomonas putida
KT2440
Sequence Analysis and Molecular
Docking of GST PP_2933 protein
with FE-Substrate complex
Protein Purification using FPLC
and SDS PAGE analysis
Heavy metal resistance and
Substrates specificities
Fig 2. Amplified Product
843 base pairs. Lane1
and 2: marker and PCR
product respectively
Fig 3. Shows SDS PAGE
indicating marker, crude and
purified protein (Lane 1, 2
and 3 respectively).
Fig 6: At 2,3 and 4 mM of FeSO4 concentration, BL21 cells with
recombinant clones show significant resistance to Fe
Fig 4. Binding affinity of GST PP_2933 with various ligands (trans-
2-octenal,1-Chloro-2,4-dinitrobenzene, dichloro-4-nitrobenzene,
iron sulphate, ethacrynic acid, cumene peroxide, epoxy-3-
nitrophenoxy propane, trans-4-phenyl-3-butane-2-one, hexa-2, 4-
dienal, dichloromethane and trans-2-hexenal respectively)
Fig 1. Multiple alignments: Representatives of the bacterial GST
classes, and putative GSTs from Pseudomonas putida KT2400.
The green shaded box indicates similarity while the red shaded
box indicates identical
 Highlighted sequence in Fig 1 is the unique N-terminal
extension of PP_2933.
 The overexpressed protein gave a thick band at expected
molecular weight of 31kDa (Fig 3).
 Preliminary enzymatic activity: PP_2933 has shown stronger
activity with ethacrynic acid (EA) and cumene peroxide when
compared with other substrates.
 Iron Sulphate, Ethacrynic Acid and Cumene peroxide have the
lowest binding energy from Molecular Docking analysis. (Fig 4)
 Lysine-100 forms hydrogen bond with Ligand-Fe complex.
While Phenylalanine-88 forms a bond with glutathione. Other
amino acids present at the active sites were Glutamine-87,
Asparagine-54 and Arginine-27. The blue color line shows
hydrogen bonds (figure 5).
 Resistance to iron concentrations might be an indication that
PP_2933 might have an FE complex binding site or Fe
complex might be involved in its stability and catalytic activity
Introduction
 Glutathione S-transferase (GST) is an enzyme that is known to participate in detoxification of
xenobiotic in several organisms.
 They catalyze conjugation between glutathione and different electrophilic substrates (Deponte,
2013).
 Less is known about the function of GST in bacteria except its ability to detoxify chlorinated
hydrocarbon.
 Here we report a unique GST (PP_2933) gene from Pseudomonas putida KT2440.
 The gene has an open reading frame of 843 base pairs encoding 280 amino acids with molecular
weight of 31.2 kDa.
 The GST was cloned, expressed and purified using ion exchange chromatography
 The purified protein shows activity with trans-2-octenal and cumene peroxide, an indication that it
might participates in oxidative stress.
 Resistance to Fe metal was observed when the recombinant GST was transformed into BL21
cells.
 Homology modelling, sequence analyses and molecular docking suggest that the GST might be a
new class of bacteria GST and could probably be a promising candidate for Fe metal
bioremediation.
CONCLUSION
 Sequence analysis revealed that PP_2933 shares 69% identity with an unusual GST yghu from E.coli.
 Yghu is a new class of bacteria GST and is termed as (Nu class), with presence of cysteine at the active site (Stourman et, al 2011).
 Both Yghu and PP_2933 possess peroxidase activity.
 We here by proposed that PP_2933 is another member of the Nu class GST (GST N3-3)
 PP_2933 does not possess cysteine in the entire structure as compared to yghu, but may utilize other amino acids for its catalytic activity.
 Further work will be to ascertain the amino acids responsible for its catalytic activity through site directed mutagenesis.
 Finally, resistance to Fe toxicity of the transformed cells is probably due to PP_2933 clone, as such the recombinant clone might be good in bioremediation of iron toxicity.
ACKNOWLEDGEMENT
Our profound gratitude goes to University of Malaya and Bayero University Kano for the research grant (PG167-2015A) and sponsorship (TETFUND) respectively.
REFERENCES
1. Deponte, M. (2013). Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes. Biochimica et Biophysica Acta (BBA)-General Subjects, 1830(5), 3217-3266.
2. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature protocols, 10(6), 845-858.
3. Stourman, N. V., Branch, M. C., Schaab, M. R., Harp, J. M., Ladner, J. E., & Armstrong, R. N. (2011). Structure and function of YghU, a nu-class glutathione transferase related to YfcG from Escherichia coli.
Biochemistry, 50(7), 1274-1281.
Minimumbindingenergy
CACC
T7Promoter GSTPP_2933
G
-
TOP10 BACTERIA CELL
EXTRACTED PLASMID
RECOMBINAN
T CLONE
PET100/D-TOPO
PCR AMPLIFIED PRODUCT 843 BASE
PAIRS
TRANSFORMATION INTO BL21 FOR PROTEIN EXPRESSION
Objectives
1. In silico analysis and homology modelling of putative GST from P . putida KT2440
2. To amplify, clone, express and purify recombinant protein (PP_2933)
3. To study the role of PP_2933 in heavy metal bioremediation
Diagram 1.
Amplification and cloning of GST PP_2933.
Cloned plasmid was transformed in to Top 10
and BL21 cells
Fig 5. Docked complex of PP_2933 with Etacrynic acid- iron complex..
Modelled structure for PP_2933 was obtained using phyre 2 software
(Kelley et al., 2015).
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2 3 4 5 6
ColonyFormingUnit
Iron sulphate concentration (mM)
Colony counts at various iron concentration
Transformed cells with recombinant plasmid Transformed cells with control plasmid
1 2 1 2 3
Substrates