Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families.
Arylmalonate-Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofa...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2016-08-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.01332/full |
| _version_ | 1811339980677578752 |
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| author | Janine Maimanakos Jennifer Chow Sarah Gaßmeyer Simon Güllert Florian Busch Robert Kourist Wolfgang R. Streit |
| author_facet | Janine Maimanakos Jennifer Chow Sarah Gaßmeyer Simon Güllert Florian Busch Robert Kourist Wolfgang R. Streit |
| author_sort | Janine Maimanakos |
| collection | DOAJ |
| description | Arylmalonate-Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta- and Gammaproteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the TTT family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes. |
| first_indexed | 2024-04-13T18:35:34Z |
| format | Article |
| id | doaj.art-d567538b076440a8b3be9bfd4ab21e1d |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-13T18:35:34Z |
| publishDate | 2016-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-d567538b076440a8b3be9bfd4ab21e1d2022-12-22T02:34:55ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2016-08-01710.3389/fmicb.2016.01332208530Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families.Janine Maimanakos0Jennifer Chow1Sarah Gaßmeyer2Simon Güllert3Florian Busch4Robert Kourist5Wolfgang R. Streit6University of HamburgUniversity of HamburgRuhr-University BochumUniversity of HamburgRuhr-University BochumRuhr-University BochumUniversity of HamburgArylmalonate-Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta- and Gammaproteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the TTT family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes.http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.01332/fullDecarboxylationFlurbiprofenABC transportersHidden Markov ModelsracemaseVariovorax sp. |
| spellingShingle | Janine Maimanakos Jennifer Chow Sarah Gaßmeyer Simon Güllert Florian Busch Robert Kourist Wolfgang R. Streit Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families. Frontiers in Microbiology Decarboxylation Flurbiprofen ABC transporters Hidden Markov Models racemase Variovorax sp. |
| title | Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families. |
| title_full | Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families. |
| title_fullStr | Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families. |
| title_full_unstemmed | Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families. |
| title_short | Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families. |
| title_sort | sequence based screening for rare enzymes new insights into the world of amdases reveal a conserved motif and 58 novel enzymes clustering in eight distinct families |
| topic | Decarboxylation Flurbiprofen ABC transporters Hidden Markov Models racemase Variovorax sp. |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.01332/full |
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