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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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Three-dimensional structure of the α2β2 tetramer of A. vinelandii Component 1 (3U7Q.pdb).The figure is centered on the approximate two-fold axis between the αβ pairs. Red is the α-subunit and blue is the β-subunit with the three metal centers shown in space filling PCK models. The Component 2 (Fe-protein) docking site is along the axis (arrow) identifying the P-cluster. Figure was prepared using Pymol (http://pymol.org/).
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pone-0072751-g001: Three-dimensional structure of the α2β2 tetramer of A. vinelandii Component 1 (3U7Q.pdb).The figure is centered on the approximate two-fold axis between the αβ pairs. Red is the α-subunit and blue is the β-subunit with the three metal centers shown in space filling PCK models. The Component 2 (Fe-protein) docking site is along the axis (arrow) identifying the P-cluster. Figure was prepared using Pymol (http://pymol.org/).

Mentions: The three dimensional structures of Components 1 and 2 as well as of several complexes between the two components have been determined for the proteins from three species including that for the Azotobacter vinelandii Component 1 at 1.0 Å [6]–[13]. Component 1 is an α2β2 tetramer of two related but different subunits where the two β subunits, β–β′, form a two-fold symmetry core with an α-subunit uniquely associated with each β-subunit, as shown in Figure 1[6], [7], [10]. Component 1 has two unique Fe:S based clusters, the 8Fe:7S P-cluster and the 7Fe:M: 9S:C:homocitrate cofactor where M can be Mo, V or another Fe atom. The P-cluster is shared at the interface of the α-β pair and can be considered two 4Fe:4S clusters fused at a common corner S with two bridging and four terminal cysteinyl ligands [14]. The cofactor, fully embedded with one in each α-subunit, is more complex having eight metals resembling the fusion of two clusters bridged by inorganic sulfides. At one corner the alternate Mo, V, or Fe atom is coordinated by a histidyl residue and the organic acid, homocitric acid. Central to the cofactor structure is an interstitial carbon atom hexacoordinated to six equidistant Fe atoms [6], [10]. Because this ensemble of the cluster and homocitric acid can be extracted intact from denatured protein, it has been called a cofactor and is abbreviated, Fe(Mo, V, or Fe) co [15]. This arrangement suggests that each α-β pair is an independent electron transfer and substrate-reducing unit. The present understanding of the reaction sequence is that electrons are transferred from the Fe-protein 4Fe:4S cluster to the P-cluster and finally to the cofactor for substrate reduction [5] (see Figure 1 for relative positions of metal centers and Component 2 binding site).


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)

Three-dimensional structure of the α2β2 tetramer of A. vinelandii Component 1 (3U7Q.pdb).The figure is centered on the approximate two-fold axis between the αβ pairs. Red is the α-subunit and blue is the β-subunit with the three metal centers shown in space filling PCK models. The Component 2 (Fe-protein) docking site is along the axis (arrow) identifying the P-cluster. Figure was prepared using Pymol (http://pymol.org/).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760896&req=5

pone-0072751-g001: Three-dimensional structure of the α2β2 tetramer of A. vinelandii Component 1 (3U7Q.pdb).The figure is centered on the approximate two-fold axis between the αβ pairs. Red is the α-subunit and blue is the β-subunit with the three metal centers shown in space filling PCK models. The Component 2 (Fe-protein) docking site is along the axis (arrow) identifying the P-cluster. Figure was prepared using Pymol (http://pymol.org/).
Mentions: The three dimensional structures of Components 1 and 2 as well as of several complexes between the two components have been determined for the proteins from three species including that for the Azotobacter vinelandii Component 1 at 1.0 Å [6]–[13]. Component 1 is an α2β2 tetramer of two related but different subunits where the two β subunits, β–β′, form a two-fold symmetry core with an α-subunit uniquely associated with each β-subunit, as shown in Figure 1[6], [7], [10]. Component 1 has two unique Fe:S based clusters, the 8Fe:7S P-cluster and the 7Fe:M: 9S:C:homocitrate cofactor where M can be Mo, V or another Fe atom. The P-cluster is shared at the interface of the α-β pair and can be considered two 4Fe:4S clusters fused at a common corner S with two bridging and four terminal cysteinyl ligands [14]. The cofactor, fully embedded with one in each α-subunit, is more complex having eight metals resembling the fusion of two clusters bridged by inorganic sulfides. At one corner the alternate Mo, V, or Fe atom is coordinated by a histidyl residue and the organic acid, homocitric acid. Central to the cofactor structure is an interstitial carbon atom hexacoordinated to six equidistant Fe atoms [6], [10]. Because this ensemble of the cluster and homocitric acid can be extracted intact from denatured protein, it has been called a cofactor and is abbreviated, Fe(Mo, V, or Fe) co [15]. This arrangement suggests that each α-β pair is an independent electron transfer and substrate-reducing unit. The present understanding of the reaction sequence is that electrons are transferred from the Fe-protein 4Fe:4S cluster to the P-cluster and finally to the cofactor for substrate reduction [5] (see Figure 1 for relative positions of metal centers and Component 2 binding site).

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
Related in: MedlinePlus