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A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

BACKGROUND: In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as...

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Autores principales: Yonemoto, Isaac T, Clarkson, Benjamin R, Smith, Hamilton O, Weyman, Philip D
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070099/
https://www.ncbi.nlm.nih.gov/pubmed/24934472
http://dx.doi.org/10.1186/1471-2091-15-10
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author Yonemoto, Isaac T
Clarkson, Benjamin R
Smith, Hamilton O
Weyman, Philip D
author_facet Yonemoto, Isaac T
Clarkson, Benjamin R
Smith, Hamilton O
Weyman, Philip D
author_sort Yonemoto, Isaac T
collection PubMed
description BACKGROUND: In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. RESULTS: Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. CONCLUSIONS: We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme.
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spelling pubmed-40700992014-06-26 A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase Yonemoto, Isaac T Clarkson, Benjamin R Smith, Hamilton O Weyman, Philip D BMC Biochem Research Article BACKGROUND: In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. RESULTS: Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. CONCLUSIONS: We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme. BioMed Central 2014-06-17 /pmc/articles/PMC4070099/ /pubmed/24934472 http://dx.doi.org/10.1186/1471-2091-15-10 Text en Copyright © 2014 Yonemoto et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/4.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Yonemoto, Isaac T
Clarkson, Benjamin R
Smith, Hamilton O
Weyman, Philip D
A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase
title A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase
title_full A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase
title_fullStr A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase
title_full_unstemmed A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase
title_short A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase
title_sort broad survey reveals substitution tolerance of residues ligating fes clusters in [nife] hydrogenase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070099/
https://www.ncbi.nlm.nih.gov/pubmed/24934472
http://dx.doi.org/10.1186/1471-2091-15-10
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