No abstract

SUMMARY Babesiosis is an emerging, tick-transmitted, zoonotic disease caused by hematotropic parasites of the genus Babesia. Babesial parasites (and those of the closely related genus Theileria) are some of the most ubiquitous and widespread blood parasites in the world, second only to the trypanosomes, and consequently have considerable worldwide economic, medical, and veterinary impact. The parasites are intraerythrocytic and are commonly called piroplasms due to the pear-shaped forms found within infected red blood cells. The piroplasms are transmitted by ixodid ticks and are capable of infecting a wide variety of vertebrate hosts which are competent in maintaining the transmission cycle. Studies involving animal hosts other than humans have contributed significantly to our understanding of the disease process, including possible pathogenic mechanisms of the parasite and immunological responses of the host. To date, there are several species of Babesia that can infect humans, Babesia microti being the most prevalent. Infections with Babesia species generally follow regional distributions; cases in the United States are caused primarily by B. microti, whereas cases in Europe are usually caused by Babesia divergens. The spectrum of disease manifestation is broad, ranging from a silent infection to a fulminant, malaria-like disease, resulting in severe hemolysis and occasionally in death. Recent advances have resulted in the development of several diagnostic tests which have increased the level of sensitivity in detection, thereby facilitating diagnosis, expediting appropriate patient management, and resulting in a more accurate epidemiological description.

Dinitrogenase, the enzyme capable of catalyzing the reduction of N2, is a heterotetramer (␣2␤2) and contains the iron-molybdenum cofactor (FeMo-co) at the active site of the enzyme. Mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase, which is enzymatically inactive but can be activated in vitro by the addition of purified FeMo-co. Apodinitrogenase from certain mutant strains of Azotobacter vinelandii has a subunit composition of ␣ 2 ␤ 2 ␥ 2 . The ␥ subunit has been implicated as necessary for the efficient activation of apodinitrogenase in vitro.Characterization of ␥ protein in crude extracts and partially pure fractions has suggested that it is a chaperone-insertase required by apodinitrogenase for the insertion of FeMo-co. There are three major forms of ␥ protein detectable by Western analysis of native gels. An apodinitrogenase-associated form is found in extracts of nifB or nifNE strains and dissociates from the apocomplex upon addition of purified FeMo-co. A second form of ␥ protein is unassociated with other proteins and exists as a homodimer. Both of these forms of ␥ protein can be converted to a third form by the addition of purified FeMo-co. This conversion requires the addition of active FeMo-co and correlates with the incorporation of iron into ␥ protein. Crude extracts that contain this form of ␥ protein are capable of donating FeMo-co to apodinitrogenase, thereby activating the apodinitrogenase. These data support a model in which ␥ protein is able to interact with both FeMo-co and apodinitrogenase, facilitate FeMo-co insertion into apodinitrogenase, and then dissociate from the activated dinitrogenase complex.Nitrogenase is comprised of two components: dinitrogenase (also known as component I or the MoFe protein) and dinitrogenase reductase (also known as component II, NifH, or the Fe protein). Nitrogenase catalyzes the ATP-and reductant-dependent reduction of N 2 and other substrates. Dinitrogenase is a 240-kDa ␣ 2 ␤ 2 tetramer encoded by nifKD (1). Dinitrogenase contains two types of metal centers: the P-cluster (8Fe-8S) (2, 3), which bridges the ␣ and ␤ subunits, and a unique ironmolybdenum cofactor (FeMo-co) 1 (4), which is buried within the ␣ subunit (3, 5) and is the site of substrate reduction (6). The dinitrogenase complex has two of each of these metal centers.Dinitrogenase reductase is a 60-kDa dimer encoded by nifH. It specifically reduces dinitrogenase, apparently transferring electrons to the P-cluster, which then channels them to FeMoco. In addition to this catalytic role, dinitrogenase reductase is also involved in the biosynthesis of FeMo-co and the maturation of dinitrogenase (reviewed in Ref.

Dinitrogenase is a heterotetrameric (␣ 2 ␤ 2 ) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition ␣ 2 ␤ 2 ␥ 2 , which can be activated in vitro by the addition of FeMo-co. The ␥ protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the ␥ subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now ␥. In vitro FeMo-co insertion experiments presented in this work demonstrate that ␥ stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter.Nitrogenase catalyzes the reduction of nitrogen gas to ammonium, in an ATP-and reductant-dependent reaction. It is one of the best characterized metalloenzymes and is an excellent model for elucidating metalloprotein assembly. Nitrogenase is composed of two oxygen-labile metalloproteins: dinitrogenase and dinitrogenase reductase (1, 2). Dinitrogenase (also termed component I or molybdenum-iron protein) is a 240-kDa ␣ 2 ␤ 2 tetramer of the nifD and nifK gene products (3). Each ␣␤ nitrogenase dimer contains an iron-molybdenum cofactor (FeMo-co) 1 and a P cluster (3, 4). Dinitrogenase reductase (also termed component II or iron protein) is a 60-kDa ␣ 2 dimer of the nifH gene product which contains a single 4Fe-4S center coordinated between the two subunits (5). NifH has at least three roles in the nitrogenase enzyme system (6): first, it serves as electron donor to nitrogenase; second, it participates in the biosynthesis of FeMo-co; and third, it is required for maturation of apodinitrogenase to a FeMo-co-activable form.