Cargando…

Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement

The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful m...

Descripción completa

Detalles Bibliográficos
Autores principales: Liu, Changshui, Wang, Qi, Xian, Mo, Ding, Yamei, Zhao, Guang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779250/
https://www.ncbi.nlm.nih.gov/pubmed/24073271
http://dx.doi.org/10.1371/journal.pone.0075554
_version_ 1782285223351812096
author Liu, Changshui
Wang, Qi
Xian, Mo
Ding, Yamei
Zhao, Guang
author_facet Liu, Changshui
Wang, Qi
Xian, Mo
Ding, Yamei
Zhao, Guang
author_sort Liu, Changshui
collection PubMed
description The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K (cat)/K (m) value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems.
format Online
Article
Text
id pubmed-3779250
institution National Center for Biotechnology Information
language English
publishDate 2013
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-37792502013-09-26 Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement Liu, Changshui Wang, Qi Xian, Mo Ding, Yamei Zhao, Guang PLoS One Research Article The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K (cat)/K (m) value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems. Public Library of Science 2013-09-20 /pmc/articles/PMC3779250/ /pubmed/24073271 http://dx.doi.org/10.1371/journal.pone.0075554 Text en © 2013 Liu et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Liu, Changshui
Wang, Qi
Xian, Mo
Ding, Yamei
Zhao, Guang
Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement
title Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement
title_full Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement
title_fullStr Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement
title_full_unstemmed Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement
title_short Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement
title_sort dissection of malonyl-coenzyme a reductase of chloroflexus aurantiacus results in enzyme activity improvement
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779250/
https://www.ncbi.nlm.nih.gov/pubmed/24073271
http://dx.doi.org/10.1371/journal.pone.0075554
work_keys_str_mv AT liuchangshui dissectionofmalonylcoenzymeareductaseofchloroflexusaurantiacusresultsinenzymeactivityimprovement
AT wangqi dissectionofmalonylcoenzymeareductaseofchloroflexusaurantiacusresultsinenzymeactivityimprovement
AT xianmo dissectionofmalonylcoenzymeareductaseofchloroflexusaurantiacusresultsinenzymeactivityimprovement
AT dingyamei dissectionofmalonylcoenzymeareductaseofchloroflexusaurantiacusresultsinenzymeactivityimprovement
AT zhaoguang dissectionofmalonylcoenzymeareductaseofchloroflexusaurantiacusresultsinenzymeactivityimprovement