Limits...
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.

Show MeSH

Related in: MedlinePlus

Sequence of the EFE-protein from P. digitatum. (A) Suggested revision of protein sequence of EFE from P. digitatum, isolate PHI26 (accession: EKV19239). In yellow shading is the present start codon of the protein EKV19239. Underlined sequence is translated from the 147 bp preceding the start codon. The sequence in red was not found in the purified EFE from P.digitatum. (B) Alignment of the sequences of P. digitatum and P. chrysogenum with the published N-terminal sequence of purified EFE from P. digitatum (indicated as: Nagahana et al.) [8]. The red X in the N-terminal sequencing is an R by homology, which fits well with the trouble of this amino acid in the chemistry of this method.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Sequence of the EFE-protein from P. digitatum. (A) Suggested revision of protein sequence of EFE from P. digitatum, isolate PHI26 (accession: EKV19239). In yellow shading is the present start codon of the protein EKV19239. Underlined sequence is translated from the 147 bp preceding the start codon. The sequence in red was not found in the purified EFE from P.digitatum. (B) Alignment of the sequences of P. digitatum and P. chrysogenum with the published N-terminal sequence of purified EFE from P. digitatum (indicated as: Nagahana et al.) [8]. The red X in the N-terminal sequencing is an R by homology, which fits well with the trouble of this amino acid in the chemistry of this method.

Mentions: The ethylene forming activity of the 2-oxoglutarate dependent route is mediated by a single enzyme termed EFE, which has been biochemically characterized from isolates of P. syringae and from P. digitatum[8]. The EFE from P. syringae pv phaseolicola is especially well studied and has been shown to mediate ethylene formation when expressed in S. cerevisiae, E. coli, T. reesii and P. putida[9-12]. Unfortunately, the gene sequence of the enzyme from P. digitatum was not known, apart from the N-terminal 25 amino acids, characterized from a purified enzyme [8]. The recently published genome sequence of P. digitatum[13] allowed us to find a protein sequence with high homology to the P. syringae EFE. However, the previously published N-terminal was only partially found in this sequence and we therefore inspected the upstream codons in the DNA-sequence. This revealed that the predicted start codon is most likely not correct. When using the first alternative start codon, which is located 147 basepairs upstream, a similar sequence to the published N-terminal was found, preceded by a stretch of amino acids predicted by the MitoProtII-software (http://ihg.gsf.de/ihg/mitoprot.html) [14] to encode a mitochondrial signal peptide (Figure 1A). The probability for mitochondrial localization was 0.9491, with a predicted cleavage site after amino acid 33 (Bold/Italic in Figure 1A). This fits well, but not perfectly, with the N-terminal identified previously [8], indicating either that the protein is further processed after cleavage or that the predicted site of cleavage is inaccurate. In further support of our change in start codon choice for the P. digitatum EFE, a highly similar protein to the protein from P. digitatum is found in Penicillium chrysogenum (accession number: XP_002562422). The P. chrysogenum protein is predicted to use a start codon at a homologous position to that which we predict in P. digitatum. It is therefore likely that the P. digitatum EFE is a mitochondrially localized protein. This is somewhat surprising in view of the fact that P. syringae EFE was found to be inactive when expressed in mitochondria of S. cerevisiae[10]. Furthermore, there was a discrepancy in the first five amino acids of the P. digitatum EFE homolog and the sequence of the purified enzyme (Figure 1B). We do not know if this reflects a technical issue of the amino acid sequencing (in which the determination of arginine, proline and threonine can be difficult) or whether it reflects the fact that the sequences are derived from two different isolates of P. digitatum. This issue, together with the fact that sequence homology does not necessarily imply equal function, made it necessary to investigate whether the EFE-homologs from Penicillium species can mediate production of ethylene. To this end we cloned and expressed the EFE - homologs from P. syringae, P. digitatum and P. chrysogenum in S. cerevisiae. The EFE-genes from Penicillium were expressed without the putative mitochondrial targeting signal. When grown in batch cultures, both the P. syringae and P. digitatum enzymes mediated ethylene production, although the P. digitatum enzyme mediated only 68% of the ethylene production seen for the P. syringae enzyme (75 ± 14 vs 111 ± 12 μg/gDW and hr, respectively; n = 2 for P. digitatum and n = 3 for P. syringae). The enzyme from P. chrysogenum produced no ethylene, despite its strong sequence similarity to the P. digitatum enzyme (data not shown).


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)

Sequence of the EFE-protein from P. digitatum. (A) Suggested revision of protein sequence of EFE from P. digitatum, isolate PHI26 (accession: EKV19239). In yellow shading is the present start codon of the protein EKV19239. Underlined sequence is translated from the 147 bp preceding the start codon. The sequence in red was not found in the purified EFE from P.digitatum. (B) Alignment of the sequences of P. digitatum and P. chrysogenum with the published N-terminal sequence of purified EFE from P. digitatum (indicated as: Nagahana et al.) [8]. The red X in the N-terminal sequencing is an R by homology, which fits well with the trouble of this amino acid in the chemistry of this method.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Sequence of the EFE-protein from P. digitatum. (A) Suggested revision of protein sequence of EFE from P. digitatum, isolate PHI26 (accession: EKV19239). In yellow shading is the present start codon of the protein EKV19239. Underlined sequence is translated from the 147 bp preceding the start codon. The sequence in red was not found in the purified EFE from P.digitatum. (B) Alignment of the sequences of P. digitatum and P. chrysogenum with the published N-terminal sequence of purified EFE from P. digitatum (indicated as: Nagahana et al.) [8]. The red X in the N-terminal sequencing is an R by homology, which fits well with the trouble of this amino acid in the chemistry of this method.
Mentions: The ethylene forming activity of the 2-oxoglutarate dependent route is mediated by a single enzyme termed EFE, which has been biochemically characterized from isolates of P. syringae and from P. digitatum[8]. The EFE from P. syringae pv phaseolicola is especially well studied and has been shown to mediate ethylene formation when expressed in S. cerevisiae, E. coli, T. reesii and P. putida[9-12]. Unfortunately, the gene sequence of the enzyme from P. digitatum was not known, apart from the N-terminal 25 amino acids, characterized from a purified enzyme [8]. The recently published genome sequence of P. digitatum[13] allowed us to find a protein sequence with high homology to the P. syringae EFE. However, the previously published N-terminal was only partially found in this sequence and we therefore inspected the upstream codons in the DNA-sequence. This revealed that the predicted start codon is most likely not correct. When using the first alternative start codon, which is located 147 basepairs upstream, a similar sequence to the published N-terminal was found, preceded by a stretch of amino acids predicted by the MitoProtII-software (http://ihg.gsf.de/ihg/mitoprot.html) [14] to encode a mitochondrial signal peptide (Figure 1A). The probability for mitochondrial localization was 0.9491, with a predicted cleavage site after amino acid 33 (Bold/Italic in Figure 1A). This fits well, but not perfectly, with the N-terminal identified previously [8], indicating either that the protein is further processed after cleavage or that the predicted site of cleavage is inaccurate. In further support of our change in start codon choice for the P. digitatum EFE, a highly similar protein to the protein from P. digitatum is found in Penicillium chrysogenum (accession number: XP_002562422). The P. chrysogenum protein is predicted to use a start codon at a homologous position to that which we predict in P. digitatum. It is therefore likely that the P. digitatum EFE is a mitochondrially localized protein. This is somewhat surprising in view of the fact that P. syringae EFE was found to be inactive when expressed in mitochondria of S. cerevisiae[10]. Furthermore, there was a discrepancy in the first five amino acids of the P. digitatum EFE homolog and the sequence of the purified enzyme (Figure 1B). We do not know if this reflects a technical issue of the amino acid sequencing (in which the determination of arginine, proline and threonine can be difficult) or whether it reflects the fact that the sequences are derived from two different isolates of P. digitatum. This issue, together with the fact that sequence homology does not necessarily imply equal function, made it necessary to investigate whether the EFE-homologs from Penicillium species can mediate production of ethylene. To this end we cloned and expressed the EFE - homologs from P. syringae, P. digitatum and P. chrysogenum in S. cerevisiae. The EFE-genes from Penicillium were expressed without the putative mitochondrial targeting signal. When grown in batch cultures, both the P. syringae and P. digitatum enzymes mediated ethylene production, although the P. digitatum enzyme mediated only 68% of the ethylene production seen for the P. syringae enzyme (75 ± 14 vs 111 ± 12 μg/gDW and hr, respectively; n = 2 for P. digitatum and n = 3 for P. syringae). The enzyme from P. chrysogenum produced no ethylene, despite its strong sequence similarity to the P. digitatum enzyme (data not shown).

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
Related in: MedlinePlus