Methanobactin transport machinery.

Methanotrophic bacteria use methane, a potent greenhouse gas, as their primary source of carbon and energy. The first step in methane metabolism is its oxidation to methanol. In almost all methanotrophs, this chemically challenging reaction is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent integral membrane enzyme. Methanotrophs acquire copper (Cu) for pMMO by secreting a small ribosomally produced, posttranslationally modified natural product called methanobactin (Mbn). Mbn chelates Cu with high affinity, and the Cu-loaded form (CuMbn) is reinternalized into the cell via an active transport process. Bioinformatic and gene regulation studies suggest that two proteins might play a role in CuMbn handling: the TonB-dependent transporter MbnT and the periplasmic binding protein MbnE. Disruption of the gene that encodes MbnT abolishes CuMbn uptake, as reported previously, and expression of MbnT in Escherichia coli confers the ability to take up CuMbn. Biophysical studies of MbnT and MbnE reveal specific interactions with CuMbn, and a crystal structure of apo MbnE is consistent with MbnE's proposed role as a periplasmic CuMbn transporter. Notably, MbnT and MbnE exhibit different levels of discrimination between cognate and noncognate CuMbns. These findings provide evidence for CuMbn-protein interactions and begin to elucidate the molecular mechanisms of its recognition and transport.

Sinorhizobium meliloti Nia is a P(1B-5)-ATPase expressed in the nodule during plant symbiosis and is involved in Ni and Fe transport.

The P1B-ATPases are a ubiquitous family of metal transporters. These transporters are classified into subfamilies on the basis of substrate specificity, which is conferred by conserved amino acids in the last three transmembrane domains. Five subfamilies have been identified to date, and representative members of four (P1B-1 to P1B-4) have been studied. The fifth family (P1B-5), of which some members contain a C-terminal hemerythrin (Hr) domain, is less well characterized. The S. meliloti Sma1163 gene encodes for a P1B-5-ATPase, denoted Nia (Nickel-iron ATPase), that is induced by exogenous Fe(2+) and Ni(2+). The nia mutant accumulates nickel and iron, suggesting a possible role in detoxification of these two elements under free-living conditions, as well as in symbiosis, when the highest expression levels are measured. This function is supported by an inhibitory effect of Fe(2+) and Ni(2+) on the pNPPase activity, and by the ability of Nia to bind Fe(2+) in the transmembrane domain. Optical and X-ray absorption spectroscopic studies of the isolated Hr domain confirm the presence of a dinuclear iron center and suggest that this domain might function as an iron sensor.

Characterization of a cobalt-specific P(1B)-ATPase.

The P(1B)-type ATPases are a ubiquitous family of P-type ATPases involved in the transport of transition metal ions. Divided into subclasses based on sequence characteristics and substrate specificity, these integral membrane transporters play key roles in metal homeostasis, metal tolerance, and the biosynthesis of metalloproteins. The P(1B-4)-ATPases have the simplest architecture of the five P(1B)-ATPase families and have been suggested to play a role in Co(2+) transport. A P(1B-4)-ATPase from Sulfitobacter sp. NAS-14.1, designated sCoaT, has been cloned, expressed, and purified. Activity assays indicate that sCoaT is specific for Co(2+). A single Co(2+) binding site is present, and optical, electron paramagnetic resonance, and X-ray absorption spectroscopic data are consistent with tetrahedral coordination by oxygen and nitrogen ligands, including a histidine and likely a water. Surprisingly, there is no evidence for coordination by sulfur. Mutation of a conserved cysteine residue, Cys 327, in the signature transmembrane Ser-Pro-Cys metal binding motif does not abolish the ATP hydrolysis activity or affect the spectroscopic analysis, establishing that this residue is not involved in the initial Co(2+) binding by sCoaT. In contrast, replacements of conserved transmembrane residues Ser 325, His 657, Glu 658, and Thr 661 with alanine abolish ATP hydrolysis activity and Co(2+) binding, indicating that these residues are necessary for Co(2+) transport. These data represent the first in vitro characterization of a P(1B-4)-ATPase and its Co(2+) binding site.