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Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton.

Yandamuri RC, Gautam R, Darkoh C, Dareddy V, El-Bouhssini M, Clack BA - Insects (2014)

Bottom Line: The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size.At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure.A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology, Stephen F. Austin State University, P.O. Box 13003, SFA Station, Nacogdoches, TX 75962, USA. yendamuri.ravi@gmail.com.

ABSTRACT
eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.

No MeSH data available.


Related in: MedlinePlus

Michaelis-Menten kinetics of the recombinant spPEP from the soluble protein fraction. PGpNA concentrations were varied from 0 to 300 µM in 0.1 M ethanolamine buffer, pH 8.5, containing 0.1 M NaCl and 1 mM DTT. The reaction was started by the addition 130 µg of purified spPEP per well. The A410 was monitored over time. The Km and Vmax were determined from the double reciprocal plot, 1/v vs 1/[GPpNA]. The inset shows the initial velocities obtained for each substrate concentration plotted vs. substrate concentration to generate the Michaelis-Menten curve. A Vmax of 106 µmole/mL/min was used with the µM concentration of PEP to determine the turnover number.
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insects-05-00762-f006: Michaelis-Menten kinetics of the recombinant spPEP from the soluble protein fraction. PGpNA concentrations were varied from 0 to 300 µM in 0.1 M ethanolamine buffer, pH 8.5, containing 0.1 M NaCl and 1 mM DTT. The reaction was started by the addition 130 µg of purified spPEP per well. The A410 was monitored over time. The Km and Vmax were determined from the double reciprocal plot, 1/v vs 1/[GPpNA]. The inset shows the initial velocities obtained for each substrate concentration plotted vs. substrate concentration to generate the Michaelis-Menten curve. A Vmax of 106 µmole/mL/min was used with the µM concentration of PEP to determine the turnover number.

Mentions: The spPEP purified from the soluble fraction exhibited a Km similar to that determined by Darkoh [2] for the enzyme purified from infested wheat. A representative Michaelis–Menten kinetics is shown in Figure 6 using zGPpNA as the substrate. The inset shows the v vs. [S] plot. The enzyme Km for the peptide substrate was determined to be 211 ± 53 µM in 25 mM ethanolamine, pH 8.5, 0.1 M NaCl, 1 mM DTT. Darkoh et al. [2] reported a Km of 65.3 µM for the enzyme purified from the infested wheat and analyzed in 25 mM ethanolamine, pH 8.5. One primary reason for the difference in Km could be the level of purity of the enzyme. The enzyme in Darkoh’s study was only partially purified, whereas the recombinant protein was greater than 95% pure, as determined from the SDS PAGE (Figure 4B). The presence of contaminating proteins might actually help in the stability and binding of the substrate to the PEP. Additional studies need to be performed, such as the addition of BSA, to the reaction to test this hypothesis. Because the enzyme was pure, the turnover number for the spPEP was able to be determined. The turnover number was 172 ± 47 µM of peptide/s/µmole of spPEP.


Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton.

Yandamuri RC, Gautam R, Darkoh C, Dareddy V, El-Bouhssini M, Clack BA - Insects (2014)

Michaelis-Menten kinetics of the recombinant spPEP from the soluble protein fraction. PGpNA concentrations were varied from 0 to 300 µM in 0.1 M ethanolamine buffer, pH 8.5, containing 0.1 M NaCl and 1 mM DTT. The reaction was started by the addition 130 µg of purified spPEP per well. The A410 was monitored over time. The Km and Vmax were determined from the double reciprocal plot, 1/v vs 1/[GPpNA]. The inset shows the initial velocities obtained for each substrate concentration plotted vs. substrate concentration to generate the Michaelis-Menten curve. A Vmax of 106 µmole/mL/min was used with the µM concentration of PEP to determine the turnover number.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592603&req=5

insects-05-00762-f006: Michaelis-Menten kinetics of the recombinant spPEP from the soluble protein fraction. PGpNA concentrations were varied from 0 to 300 µM in 0.1 M ethanolamine buffer, pH 8.5, containing 0.1 M NaCl and 1 mM DTT. The reaction was started by the addition 130 µg of purified spPEP per well. The A410 was monitored over time. The Km and Vmax were determined from the double reciprocal plot, 1/v vs 1/[GPpNA]. The inset shows the initial velocities obtained for each substrate concentration plotted vs. substrate concentration to generate the Michaelis-Menten curve. A Vmax of 106 µmole/mL/min was used with the µM concentration of PEP to determine the turnover number.
Mentions: The spPEP purified from the soluble fraction exhibited a Km similar to that determined by Darkoh [2] for the enzyme purified from infested wheat. A representative Michaelis–Menten kinetics is shown in Figure 6 using zGPpNA as the substrate. The inset shows the v vs. [S] plot. The enzyme Km for the peptide substrate was determined to be 211 ± 53 µM in 25 mM ethanolamine, pH 8.5, 0.1 M NaCl, 1 mM DTT. Darkoh et al. [2] reported a Km of 65.3 µM for the enzyme purified from the infested wheat and analyzed in 25 mM ethanolamine, pH 8.5. One primary reason for the difference in Km could be the level of purity of the enzyme. The enzyme in Darkoh’s study was only partially purified, whereas the recombinant protein was greater than 95% pure, as determined from the SDS PAGE (Figure 4B). The presence of contaminating proteins might actually help in the stability and binding of the substrate to the PEP. Additional studies need to be performed, such as the addition of BSA, to the reaction to test this hypothesis. Because the enzyme was pure, the turnover number for the spPEP was able to be determined. The turnover number was 172 ± 47 µM of peptide/s/µmole of spPEP.

Bottom Line: The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size.At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure.A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology, Stephen F. Austin State University, P.O. Box 13003, SFA Station, Nacogdoches, TX 75962, USA. yendamuri.ravi@gmail.com.

ABSTRACT
eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.

No MeSH data available.


Related in: MedlinePlus