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Published September 3, 2013 | Supplemental Material + Published
Journal Article Open

Multiple Amino Acid Sequence Alignment Nitrogenase Component 1: Insights into Phylogenetics and Structure-Function Relationships


Amino acid residues critical for a protein's structure-function are retained by natural selection and these residues are identified by the level of variance in co-aligned homologous protein sequences. The relevant residues in the nitrogen fixation Component 1 α- and β-subunits were identified by the alignment of 95 protein sequences. Proteins were included from species encompassing multiple microbial phyla and diverse ecological niches as well as the nitrogen fixation genotypes, anf, nif, and vnf, which encode proteins associated with cofactors differing at one metal site. After adjusting for differences in sequence length, insertions, and deletions, the remaining >85% of the sequence co-aligned the subunits from the three genotypes. Six Groups, designated Anf, Vnf , and Nif I-IV, were assigned based upon genetic origin, sequence adjustments, and conserved residues. Both subunits subdivided into the same groups. Invariant and single variant residues were identified and were defined as "core" for nitrogenase function. Three species in Group Nif-III, Candidatus Desulforudis audaxviator, Desulfotomaculum kuznetsovii, and Thermodesulfatator indicus, were found to have a seleno-cysteine that replaces one cysteinyl ligand of the 8Fe:7S, P-cluster. Subsets of invariant residues, limited to individual groups, were identified; these unique residues help identify the gene of origin (anf, nif, or vnf) yet should not be considered diagnostic of the metal content of associated cofactors. Fourteen of the 19 residues that compose the cofactor pocket are invariant or single variant; the other five residues are highly variable but do not correlate with the putative metal content of the cofactor. The variable residues are clustered on one side of the cofactor, away from other functional centers in the three dimensional structure. Many of the invariant and single variant residues were not previously recognized as potentially critical and their identification provides the bases for new analyses of the three-dimensional structure and for mutagenesis studies.

Additional Information

© 2013 Howard et al. 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 credited. Received June 17, 2013; Accepted July 18, 2013; Published September 3, 2013. This work was supported by National Institutes of Health Grant GM45162 to DCR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: Conceived and designed the experiments: JBH KJK DCR ANG. Analyzed the data: JBH KJK DCR ANG. Wrote the paper: JBH KJK DCR ANG.

Attached Files

Published - journal.pone.0072751.pdf

Supplemental Material - journal.pone.0072751.s001.pdf

Supplemental Material - journal.pone.0072751.s002.pdf

Supplemental Material - journal.pone.0072751.s003.pdf

Supplemental Material - journal.pone.0072751.s004.pdf

Supplemental Material - journal.pone.0072751.s005.pdf

Supplemental Material - journal.pone.0072751.s006.pdf

Supplemental Material - journal.pone.0072751.s007.pdf

Supplemental Material - journal.pone.0072751.s008.pdf

Supplemental Material - journal.pone.0072751.s009.pdf

Supplemental Material - journal.pone.0072751.s010.pdf

Supplemental Material - journal.pone.0072751.s011.pdf


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