Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs
For the multimeric enzymes R-ω-transaminase (RTA), alanine dehydrogenase (AlaDH), and formate dehydrogenase (FDH), peptide bond formation between the hetrosubunits has been achieved by the intein-mediated in vivo subunit splicing. The subunit ligation is triggered by the heterodimerization of an arg...
Main Authors: | , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Frontiers Media S.A.
2020-06-01
|
Series: | Frontiers in Bioengineering and Biotechnology |
Subjects: | |
Online Access: | https://www.frontiersin.org/article/10.3389/fbioe.2020.00686/full |
id |
doaj-475976734a634dff89ec8b69de56e51b |
---|---|
record_format |
Article |
spelling |
doaj-475976734a634dff89ec8b69de56e51b2020-11-25T03:17:10ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852020-06-01810.3389/fbioe.2020.00686544004Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper MotifsRong LiYao ChenKun DuWei FengFor the multimeric enzymes R-ω-transaminase (RTA), alanine dehydrogenase (AlaDH), and formate dehydrogenase (FDH), peptide bond formation between the hetrosubunits has been achieved by the intein-mediated in vivo subunit splicing. The subunit ligation is triggered by the heterodimerization of an arginine rich leucine zipper motif with a glutamic acid rich leucine zipper motif. The one-by-one ligation of hetrosubunits constructs the pairing enzymes RTA&AlaDH and AlaDH&FDH. The ligation modes were analyzed based on blue native polyacrylamide gel electrophoresis (BN-PAGE). The spectra of circular dichroism (CD), fluorescence, and two-dimensional FTIR provide information on the secondary structures and stability of the pairing enzymes. The enzyme-substrate interaction was analyzed based on microscale thermophoresis analysis. In contrast to the mixed three enzymes RTA + AlaDH + FDH, the ligated enzymes RTA&AlaDH + AlaDH&FDH exhibited a much larger substrate affinity, higher stability, and significantly enhanced activity.https://www.frontiersin.org/article/10.3389/fbioe.2020.00686/fullR-ω-transaminasealanine dehydrogenaseformate dehydrogenasehetrosubunit splicingleucine zipper motifs |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Rong Li Yao Chen Kun Du Wei Feng |
spellingShingle |
Rong Li Yao Chen Kun Du Wei Feng Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs Frontiers in Bioengineering and Biotechnology R-ω-transaminase alanine dehydrogenase formate dehydrogenase hetrosubunit splicing leucine zipper motifs |
author_facet |
Rong Li Yao Chen Kun Du Wei Feng |
author_sort |
Rong Li |
title |
Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs |
title_short |
Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs |
title_full |
Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs |
title_fullStr |
Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs |
title_full_unstemmed |
Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs |
title_sort |
peptide bond formation between the hetrosubunits of ω-transaminase, alanine dehydrogenase, and formate dehydrogenase through subunit splicing promoted by heterodimerization of leucine zipper motifs |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Bioengineering and Biotechnology |
issn |
2296-4185 |
publishDate |
2020-06-01 |
description |
For the multimeric enzymes R-ω-transaminase (RTA), alanine dehydrogenase (AlaDH), and formate dehydrogenase (FDH), peptide bond formation between the hetrosubunits has been achieved by the intein-mediated in vivo subunit splicing. The subunit ligation is triggered by the heterodimerization of an arginine rich leucine zipper motif with a glutamic acid rich leucine zipper motif. The one-by-one ligation of hetrosubunits constructs the pairing enzymes RTA&AlaDH and AlaDH&FDH. The ligation modes were analyzed based on blue native polyacrylamide gel electrophoresis (BN-PAGE). The spectra of circular dichroism (CD), fluorescence, and two-dimensional FTIR provide information on the secondary structures and stability of the pairing enzymes. The enzyme-substrate interaction was analyzed based on microscale thermophoresis analysis. In contrast to the mixed three enzymes RTA + AlaDH + FDH, the ligated enzymes RTA&AlaDH + AlaDH&FDH exhibited a much larger substrate affinity, higher stability, and significantly enhanced activity. |
topic |
R-ω-transaminase alanine dehydrogenase formate dehydrogenase hetrosubunit splicing leucine zipper motifs |
url |
https://www.frontiersin.org/article/10.3389/fbioe.2020.00686/full |
work_keys_str_mv |
AT rongli peptidebondformationbetweenthehetrosubunitsofōtransaminasealaninedehydrogenaseandformatedehydrogenasethroughsubunitsplicingpromotedbyheterodimerizationofleucinezippermotifs AT yaochen peptidebondformationbetweenthehetrosubunitsofōtransaminasealaninedehydrogenaseandformatedehydrogenasethroughsubunitsplicingpromotedbyheterodimerizationofleucinezippermotifs AT kundu peptidebondformationbetweenthehetrosubunitsofōtransaminasealaninedehydrogenaseandformatedehydrogenasethroughsubunitsplicingpromotedbyheterodimerizationofleucinezippermotifs AT weifeng peptidebondformationbetweenthehetrosubunitsofōtransaminasealaninedehydrogenaseandformatedehydrogenasethroughsubunitsplicingpromotedbyheterodimerizationofleucinezippermotifs |
_version_ |
1724633094801063936 |