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Altered nitrogenase MoFe proteins from Azotobacter vinelandii. Analysis of MoFe proteins having amino acid substitutions for the conserved cysteine residues within the beta-subunit.

ABSTRACT: The regions surrounding the three strictly conserved cysteine residues (positions 70, 95 and 153) in the beta-subunit of the Azotobacter vinelandii nitrogenase MoFe protein have been proposed to provide P-cluster environments [Dean, Setterquist, Brigle, Scott, Laird & Newton (1990) Mol. Microbiol. 4, 1505-1512]. In the present study, each of these cysteine residues was individually substituted by either serine or alanine by site-directed mutagenesis of the nifK gene, which encodes the MoFe protein beta-subunit. A mutant strain for which the codon for Cys-153 is removed was also isolated. Significant structural or functional roles are indicated for the cysteine residues at positions 70 and 95, where substitution by either serine or alanine eliminates diazotrophic growth of the resulting strains and abolishes or markedly decreases both MoFe-protein acetylene-reduction activity and the intensity of the whole-cell S = 3/2 e.p.r. signal. Changes introduced at position 153 have various effects on the functional properties of the enzyme. The strains produced either by deletion of the Cys-153 residue or its substitution by serine exhibit only a moderate decrease in diazotrophic growth and MoFe-protein activity and no loss of the whole-cell e.p.r.-signal intensity. In contrast, substitution by alanine eliminates diazotrophic growth and very markedly decreases both MoFe-protein activity and e.p.r.-signal intensity. These results are interpreted in terms of a metallocluster-driven protein rearrangement. After purification of the altered MoFe protein, in which serine replaces Cys-153, an investigation of its catalytic and spectroscopic properties confirms that neither the FeMo cofactor, i.e. the substrate-reduction site, nor the component-protein interaction site has been affected. Instead, these data indicate a disruption in electron transfer within the MoFe protein, which is consistent with a role for this residue (and region) at the P clusters.

SUBMITTER: May HD

PROVIDER: S-EPMC1151256 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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Project description:Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

Project description:The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site. IMPORTANCE Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.

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Altered nitrogenase MoFe proteins from Azotobacter vinelandii. Analysis of MoFe proteins having amino acid substitutions for the conserved cysteine residues within the beta-subunit.

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