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Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor.

ABSTRACT: Nitrogenase-3 of Azotobacter vinelandii is synthesized under conditions of molybdenum and vanadium deficiency. The minimal metal requirement for its synthesis, and its metal content, indicated that the only transition metal in nitrogenase-3 was iron [Chisnell, Premakumar and Bishop (1988) J. Bacteriol. 170, 27-33; Pau, Mitchenall and Robson (1989) J. Bacteriol. 171, 124-129]. A new species of nitrogenase-3 has been purified from a strain of A. vinelandii (RP306) lacking structural genes for the Mo- and V-nitrogenases and containing a mutation which enables nitrogenase-3 to be synthesized in the presence of molybdenum. SDS/PAGE showed that component 1 contained a 15 kDa polypeptide which N-terminal amino acid sequence determination showed to be encoded by anfG. This confirms that nitrogenase-3, like V-nitrogenase, comprises three subunits. Preparations of the nitrogenase-3 from strain RP306 contained 24 Fe atoms and 1 Mo atom per molecule. Characterization of the cofactor centre of the enzyme by e.p.r. spectroscopy and an enzymic cofactor assay, together with stimulation of the growth of strain RP306 by Mo, showed that nitrogenase-3 can incorporate the Mo-nitrogenase cofactor (FeMoco) to form a functional enzyme. The specific activities (nmol of product produced/min per mg of protein) determined from activity titration curves were: under N2, NH3 formation 110, with concomitant H2 evolution of 220; under argon, H2 evolution 350; under 10% acetylene (C2H2) in argon, ethylene (C2H4) 58, ethane (C2H6) 26, and concomitant H2 evolution 226. The rate of formation of C2H6 was non-linear, and the C2H6/C2H4 ratio strongly dependent on the ratio of nitrogenase components.

SUBMITTER: Pau RN

PROVIDER: S-EPMC1134325 | biostudies-other | 1993 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.

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Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor.

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