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Multiple amino acid sequence alignment nitrogenase component 1: insights into phylogenetics and structure-function relationships.

Howard JB, Kechris KJ, Rees DC, Glazer AN - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America ; Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.

ABSTRACT
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.

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Cofactor environment showing amino acid residues at 5 Å contact.Cofactor including homocitric acid, α-His442, and α-Cys 275 ligands are shown as CPK spheres. Waters are red dot spheres. Dark green surface and sticks represent invariant residues. Light teal surface and sticks represent single variant residues. Bright orange surface and sticks represent multiple variant residues. (See Tables S9 and S10) A. Cofactor with α-Cys275 and α-His442 ligands. B. Invariant and single variant residues added. C Multiple variant residues added. Water sheet between homocitric acid and β-subunit is on the right. Amino acids that interact only by H-bond through water atoms are omitted. Figure was prepared using 3U7Q.pdb and Pymol (http://pymol.org/).
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pone-0072751-g004: Cofactor environment showing amino acid residues at 5 Å contact.Cofactor including homocitric acid, α-His442, and α-Cys 275 ligands are shown as CPK spheres. Waters are red dot spheres. Dark green surface and sticks represent invariant residues. Light teal surface and sticks represent single variant residues. Bright orange surface and sticks represent multiple variant residues. (See Tables S9 and S10) A. Cofactor with α-Cys275 and α-His442 ligands. B. Invariant and single variant residues added. C Multiple variant residues added. Water sheet between homocitric acid and β-subunit is on the right. Amino acids that interact only by H-bond through water atoms are omitted. Figure was prepared using 3U7Q.pdb and Pymol (http://pymol.org/).

Mentions: The cofactor environment can be divided into two parts determined by areas around the metal cluster or around the homocitric acid portions. The cluster environment appears to be more highly conserved as indicated in Table S9, where 14 of 19 residues across all six groups are invariant (9) or highly similar, single variant (5) residues. Within each of the six Groups, the residues around the cluster have a higher degree of conservation–higher fraction of invariant residues–than for the full 95 sequences. However, most significantly, there does not appear to be any obvious correlation of amino acid variants to the gene of origin (nif, anf, or vnf) or to the absence of the ancillary NifE/N proteins (see discussion above). A detailed structural analysis revealed that the most highly variable residues are not randomly distributed around the cofactor metal cluster but are concentrated on one face as shown in Figure 4. This face containing the hyper-variable residues is towards, though not on, the surface of the protein, e.g., variable α-Leu-358 is partially exposed to solvent prior to cofactor insertion [59]. The highly conserved, invariant and single variant residues on the other faces are directed towards the P-cluster. Several of these residues previously have been probed by site specific mutagenesis and have been shown to alter the cofactor spectral properties and substrate specificity, e.g., α-Val70, α-Arg96, and α-His195 [56], [57] which further emphasizes the importance of the conserved residues around the cofactor in substrate binding and electron transfer.


Multiple amino acid sequence alignment nitrogenase component 1: insights into phylogenetics and structure-function relationships.

Howard JB, Kechris KJ, Rees DC, Glazer AN - PLoS ONE (2013)

Cofactor environment showing amino acid residues at 5 Å contact.Cofactor including homocitric acid, α-His442, and α-Cys 275 ligands are shown as CPK spheres. Waters are red dot spheres. Dark green surface and sticks represent invariant residues. Light teal surface and sticks represent single variant residues. Bright orange surface and sticks represent multiple variant residues. (See Tables S9 and S10) A. Cofactor with α-Cys275 and α-His442 ligands. B. Invariant and single variant residues added. C Multiple variant residues added. Water sheet between homocitric acid and β-subunit is on the right. Amino acids that interact only by H-bond through water atoms are omitted. Figure was prepared using 3U7Q.pdb and Pymol (http://pymol.org/).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760896&req=5

pone-0072751-g004: Cofactor environment showing amino acid residues at 5 Å contact.Cofactor including homocitric acid, α-His442, and α-Cys 275 ligands are shown as CPK spheres. Waters are red dot spheres. Dark green surface and sticks represent invariant residues. Light teal surface and sticks represent single variant residues. Bright orange surface and sticks represent multiple variant residues. (See Tables S9 and S10) A. Cofactor with α-Cys275 and α-His442 ligands. B. Invariant and single variant residues added. C Multiple variant residues added. Water sheet between homocitric acid and β-subunit is on the right. Amino acids that interact only by H-bond through water atoms are omitted. Figure was prepared using 3U7Q.pdb and Pymol (http://pymol.org/).
Mentions: The cofactor environment can be divided into two parts determined by areas around the metal cluster or around the homocitric acid portions. The cluster environment appears to be more highly conserved as indicated in Table S9, where 14 of 19 residues across all six groups are invariant (9) or highly similar, single variant (5) residues. Within each of the six Groups, the residues around the cluster have a higher degree of conservation–higher fraction of invariant residues–than for the full 95 sequences. However, most significantly, there does not appear to be any obvious correlation of amino acid variants to the gene of origin (nif, anf, or vnf) or to the absence of the ancillary NifE/N proteins (see discussion above). A detailed structural analysis revealed that the most highly variable residues are not randomly distributed around the cofactor metal cluster but are concentrated on one face as shown in Figure 4. This face containing the hyper-variable residues is towards, though not on, the surface of the protein, e.g., variable α-Leu-358 is partially exposed to solvent prior to cofactor insertion [59]. The highly conserved, invariant and single variant residues on the other faces are directed towards the P-cluster. Several of these residues previously have been probed by site specific mutagenesis and have been shown to alter the cofactor spectral properties and substrate specificity, e.g., α-Val70, α-Arg96, and α-His195 [56], [57] which further emphasizes the importance of the conserved residues around the cofactor in substrate binding and electron transfer.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America ; Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.

ABSTRACT
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.

Show MeSH