VnfY Is Required for Full Activity of the Vanadium-Containing Dinitrogenase in Azotobacter vinelandii

Abstract

A gene from Azotobacter vinelandii whose product exhibits primary sequence similarity to the NifY, NafY, NifX, and VnfX family of proteins, and which is required for effective V-dependent diazotrophic growth, was identified. Because this gene is located downstream from vnfK in an arrangement similar to the relative organization of the nifK and nifY genes, it was designated vnfY. A mutant strain having an insertion mutation in vnfY has 10-fold less vnf dinitrogenase activity and exhibits a greatly diminished level of 49 V label incorporation into the V-dependent dinitrogenase when compared to the wild type. These results indicate that VnfY has a role in the maturation of the V-dependent dinitrogenase, with a specific role in the formation of the V-containing cofactor and/or its insertion into apodinitrogenase.Azotobacter vinelandii harbors three genetically distinct nitrogenase systems that are differentially expressed depending on the availability of metals in the medium: a nif-encoded Mo-containing nitrogenase, a vnf-encoded V-containing nitrogenase, and an anf-encoded iron-only nitrogenase (2). The V-containing nitrogenase contains an iron-vanadium cofactor (FeV-co) at its active site which is structurally and functionally analogous to the better-characterized FeMo-co of the Modependent system. A functional V-containing nitrogenase requires the products of the structural genes for dinitrogenase (vnfDGK) and dinitrogenase reductase (vnfH) as well as several other nif and vnf gene products involved in the biosynthesis of FeV-co and the maturation of the nitrogenase component proteins. Much of what is known about the biosynthesis of FeV-co comes from analogous studies on FeMo-co biosynthesis. It is believed that the products of nifB, vnfN, vnfE, vnfH, vnfX, and nifV are involved in the biosynthesis of FeV-co (see reference 12 for a review).

Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii

“…2B; also see Table S2 in the supplemental material). Type IV pili are involved in a number of processes, including cell motility, DNA uptake, sensing, attachment, and aggregation (55); the latter could be a previously unrecognized O 2 protection mechanism in A. vinelandii.…”

“…2A), paralogs of NifX and NifY. VnfY is required for optimal V-dependent diazotrophy (55), and large increases in vnfX and vnfY transcript levels (15-and 117-fold) were observed during V-dependent diazotrophy (Fig. 3A).…”

Maturation of Nitrogenase: a Biochemical Puzzle

“…A number of vnf genes (named for vanadium nitrogen fixation) have been implicated in the biosynthesis of FeV-co (vnfN, vnfE, vnfX, and vnfH) and in the maturation of the VFe protein (vnfG and vnfY) in A. vinelandii (Table 2) (56,70). Very little is known about the role of anf-specific genes (named for alternative nitrogen fixation) in the biosynthesis of FeFe-co.…”

“…In fact, NifY and NafY exhibit amino acid sequence similarity to each other and are part of a family of small proteins, which we propose to name the NifX family of proteins, and whose common role seems to be the carriage of FeMo-co, FeV-co, and their biosynthetic precursors for cofactor assembly and insertion (Table 3) (20,33,55,(69)(70)(71). In A. vinelandii, some of the proteins from the NifX family exhibit a certain degree of cross-functionality in vivo (68).…”

Tungsten, the Surprisingly Positively Acting Heavy Metal Element for Prokaryotes

“…However, interest in the various Fe‐Mo‐cofactor–containing nitrogenases is still high due to the large economic impact of dinitrogen fixation to ammonia and the biochemical puzzle of its maturation, 31 despite its generally observed extreme lability as an isolated enzyme. Few bacteria, such as Azotobacter vinelandii , are actually able to form three genetically distinct and active nitrogenases, depending on the availability of molybdenum ( nif ), vanadium ( vnf ), or iron‐only ( anf ) 32 . The tungsten‐substituted form is inactive in reducing dinitrogen 33 .…”