Classifying the metal dependence of uncharacterized nitrogenases

Abstract

Nitrogenase enzymes have evolved complex iron-sulfur (Fe-S) containing cofactors that most commonly contain molybdenum (MoFe, Nif) as a heterometal but also exist as vanadium (VFe, Vnf) and heterometal-independent (Fe-only, Anf) forms. All three varieties are capable of the reduction of dinitrogen (N(2)) to ammonia (NH(3)) but exhibit differences in catalytic rates and substrate specificity unique to metal type. Recently, N(2) reduction activity was observed in archaeal methanotrophs and methanogens that encode for nitrogenase homologs which do not cluster phylogenetically with previously characterized nitrogenases. To gain insight into the metal cofactors of these uncharacterized nitrogenase homologs, predicted three-dimensional structures of the nitrogenase active site metal-cofactor binding subunits NifD, VnfD, and AnfD were generated and compared. Dendrograms based on structural similarity indicate nitrogenase homologs cluster based on heterometal content and that uncharacterized nitrogenase D homologs cluster with NifD, providing evidence that the structure of the enzyme has evolved in response to metal utilization. Characterization of the structural environment of the nitrogenase active site revealed amino acid variations that are unique to each class of nitrogenase as defined by heterometal cofactor content; uncharacterized nitrogenases contain amino acids near the active site most similar to NifD. Together, these results suggest that uncharacterized nitrogenase homologs present in numerous anaerobic methanogens, archaeal methanotrophs, and firmicutes bind FeMo-co in their active site, and add to growing evidence that diversification of metal utilization likely occurred in an anoxic habitat.

Keywords: iron; metalloenzyme; molecular similarity; molybdenum; nitrogenase; sequence conservation; vanadium.

FIGURE 1

The active site of nitrogenase from A.vinelandii (Einsle et al., 2002; Lancaster et al., 2011; Spatzal et al., 2011). The metal cofactor–homocitrate complex is shown as balls and sticks and the residues found to be within 5 Å of the cofactor are shown as lines (thin). Colors: light blue, carbon; blue, nitrogen; red, oxygen; yellow, sulfur; rust, iron; cyan, molybdenum.

FIGURE 2

Maximum likelihood unrooted phylogram of nitrogenase sequences. FeMo-co NifD-A and NifD-B sequences in black and orange, respectively, uncharacterized nitrogenases in purple, AnfD in red, and VnfD in green.

FIGURE 3

Cartoon diagram of the A.vinelandii nitrogenase D subunits. The NifD structure, drawn from 1M1N is in black (A), and predicted structures for the A. vinelandii vanadium (VnfD) and Fe-only (AnfD) homologs are in green (B) and red (C), respectively. A structural superposition of the three as performed in PyMol is shown in (D).

FIGURE 4

Structural relationships among nitrogenase D subunits as calculated by Voronoi contacts and the complete linkage clustering method. NifD-A are denoted in black, NifD-B in orange, uncharacterized nitrogenases in purple, AnfD in red, and VnfD in green.