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Comparative sequence analysis and mutagenesis of ethylene forming enzyme (EFE) 2-oxoglutarate/Fe(II)-dependent dioxygenase homologs.

Johansson N, Persson KO, Larsson C, Norbeck J - BMC Biochem. (2014)

Bottom Line: The sequence of the EFE homologs from P.digitatum and P. syringae was compared to that of the non-functional EFE-homolog from Penicillium chrysogenum and ten amino acids were found to correlate with ethylene production.Several of these amino acid residues were found to be important for ethylene production via point mutations in P. syringae EFE.We conclude that residues in addition to the 10 identified positions implicated in ethylene production by sequence comparison, are important for determining ethylene formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden. norbeck@chalmers.se.

ABSTRACT

Background: Ethylene is one of the most used chemical monomers derived from non-renewable sources and we are investigating the possibility of producing it in yeast via the ethylene forming enzyme (EFE) from Pseudomonas syringae. To enable engineering strategies to improve the enzyme, it is necessary to identify the regions and amino acid residues involved in ethylene formation.

Results: We identified the open reading frame for the EFE homolog in Penicillium digitatum and also showed its capability of mediating ethylene production in yeast. The sequence of the EFE homologs from P.digitatum and P. syringae was compared to that of the non-functional EFE-homolog from Penicillium chrysogenum and ten amino acids were found to correlate with ethylene production. Several of these amino acid residues were found to be important for ethylene production via point mutations in P. syringae EFE. The EFE homolog from P. chrysogenum was engineered at 10 amino acid residues to mimic the P. syringae EFE, but this did not confer ethylene producing capability.Furthermore, we predicted the structure of EFE by homology to known structures of 2-oxoglutarate/Fe(II) dependent dioxygenases. Three of the amino acids correlating with ethylene production are located in the predicted 2-oxoglutarate binding domain. A protein domain specific for the EFE-class was shown to be essential for activity. Based on the structure and alanine substitutions, it is likely that amino acids (H189, D191 and H268) are responsible for binding the Fe(II) ligand.

Conclusion: We provide further insight into the structure and function of the ethylene forming (EFE) - subclass of 2-oxoglutarate/Fe(II) dependent dioxygenases. We conclude that residues in addition to the 10 identified positions implicated in ethylene production by sequence comparison, are important for determining ethylene formation. We also demonstrate the use of an alternative EFE gene. The data from this study will provide the basis for directed protein engineering to enhance the ethylene production capability and properties of EFE.

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ClustalOmega-alignment of the amino acid sequences of EFE-homologs from P. syringae, P. digitatum and P. chrysogenum. Amino acids in yellow shading correlate with ethylene production. Amino acids in blue are not included in the predicted structural model. Green shaded amino acids are predicted to bind the Fe(II), the blue shaded histidines were previously suggested to bind Fe(II). Amino acids in red are predicted to bind 2-oxoglutarate and correspond to the EFE-site I [6]. Putative mitochondrial targeting sequences are marked in grey.
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Figure 2: ClustalOmega-alignment of the amino acid sequences of EFE-homologs from P. syringae, P. digitatum and P. chrysogenum. Amino acids in yellow shading correlate with ethylene production. Amino acids in blue are not included in the predicted structural model. Green shaded amino acids are predicted to bind the Fe(II), the blue shaded histidines were previously suggested to bind Fe(II). Amino acids in red are predicted to bind 2-oxoglutarate and correspond to the EFE-site I [6]. Putative mitochondrial targeting sequences are marked in grey.

Mentions: Aligning of the sequences from the two ethylene producing homologs of EFE and the ethylene production negative homolog from P. chrysogenum, using ClustalOmega [15], subsequently allowed us to assign amino acids correlating with ethylene forming ability of the enzyme. Ten residues came out as promising targets, i.e. being conserved in P. syringae and P. digitatum but non-conserved in P. chrysogenum (Figure 2, yellow shading). In order to test the hypothesis that amino acids at these ten positions would be important for ethylene production, we made point mutations on all ten positions, i.e. L22M, V172T, A199G, V212Y/E213S, E235D, I254M, F278Y, I304N and I322V, thereby changing the amino acid to those found in the ethylene production negative EFE-homolog from P. chrysogenum. The A199G, I304N and V212Y/E213S mutants all showed a clear reduction of ethylene production, while L22M, V172T, E235D, I254M, F278Y and I322V did not cause a significant change in ethylene production (Figure 3).


Comparative sequence analysis and mutagenesis of ethylene forming enzyme (EFE) 2-oxoglutarate/Fe(II)-dependent dioxygenase homologs.

Johansson N, Persson KO, Larsson C, Norbeck J - BMC Biochem. (2014)

ClustalOmega-alignment of the amino acid sequences of EFE-homologs from P. syringae, P. digitatum and P. chrysogenum. Amino acids in yellow shading correlate with ethylene production. Amino acids in blue are not included in the predicted structural model. Green shaded amino acids are predicted to bind the Fe(II), the blue shaded histidines were previously suggested to bind Fe(II). Amino acids in red are predicted to bind 2-oxoglutarate and correspond to the EFE-site I [6]. Putative mitochondrial targeting sequences are marked in grey.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4202696&req=5

Figure 2: ClustalOmega-alignment of the amino acid sequences of EFE-homologs from P. syringae, P. digitatum and P. chrysogenum. Amino acids in yellow shading correlate with ethylene production. Amino acids in blue are not included in the predicted structural model. Green shaded amino acids are predicted to bind the Fe(II), the blue shaded histidines were previously suggested to bind Fe(II). Amino acids in red are predicted to bind 2-oxoglutarate and correspond to the EFE-site I [6]. Putative mitochondrial targeting sequences are marked in grey.
Mentions: Aligning of the sequences from the two ethylene producing homologs of EFE and the ethylene production negative homolog from P. chrysogenum, using ClustalOmega [15], subsequently allowed us to assign amino acids correlating with ethylene forming ability of the enzyme. Ten residues came out as promising targets, i.e. being conserved in P. syringae and P. digitatum but non-conserved in P. chrysogenum (Figure 2, yellow shading). In order to test the hypothesis that amino acids at these ten positions would be important for ethylene production, we made point mutations on all ten positions, i.e. L22M, V172T, A199G, V212Y/E213S, E235D, I254M, F278Y, I304N and I322V, thereby changing the amino acid to those found in the ethylene production negative EFE-homolog from P. chrysogenum. The A199G, I304N and V212Y/E213S mutants all showed a clear reduction of ethylene production, while L22M, V172T, E235D, I254M, F278Y and I322V did not cause a significant change in ethylene production (Figure 3).

Bottom Line: The sequence of the EFE homologs from P.digitatum and P. syringae was compared to that of the non-functional EFE-homolog from Penicillium chrysogenum and ten amino acids were found to correlate with ethylene production.Several of these amino acid residues were found to be important for ethylene production via point mutations in P. syringae EFE.We conclude that residues in addition to the 10 identified positions implicated in ethylene production by sequence comparison, are important for determining ethylene formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden. norbeck@chalmers.se.

ABSTRACT

Background: Ethylene is one of the most used chemical monomers derived from non-renewable sources and we are investigating the possibility of producing it in yeast via the ethylene forming enzyme (EFE) from Pseudomonas syringae. To enable engineering strategies to improve the enzyme, it is necessary to identify the regions and amino acid residues involved in ethylene formation.

Results: We identified the open reading frame for the EFE homolog in Penicillium digitatum and also showed its capability of mediating ethylene production in yeast. The sequence of the EFE homologs from P.digitatum and P. syringae was compared to that of the non-functional EFE-homolog from Penicillium chrysogenum and ten amino acids were found to correlate with ethylene production. Several of these amino acid residues were found to be important for ethylene production via point mutations in P. syringae EFE. The EFE homolog from P. chrysogenum was engineered at 10 amino acid residues to mimic the P. syringae EFE, but this did not confer ethylene producing capability.Furthermore, we predicted the structure of EFE by homology to known structures of 2-oxoglutarate/Fe(II) dependent dioxygenases. Three of the amino acids correlating with ethylene production are located in the predicted 2-oxoglutarate binding domain. A protein domain specific for the EFE-class was shown to be essential for activity. Based on the structure and alanine substitutions, it is likely that amino acids (H189, D191 and H268) are responsible for binding the Fe(II) ligand.

Conclusion: We provide further insight into the structure and function of the ethylene forming (EFE) - subclass of 2-oxoglutarate/Fe(II) dependent dioxygenases. We conclude that residues in addition to the 10 identified positions implicated in ethylene production by sequence comparison, are important for determining ethylene formation. We also demonstrate the use of an alternative EFE gene. The data from this study will provide the basis for directed protein engineering to enhance the ethylene production capability and properties of EFE.

Show MeSH