Bioenergetics of archaea: ATP synthesis under harsh environmental conditions.

Archaea are a heterogeneous group of microorganisms that often thrive under harsh environmental conditions such as high temperatures, extreme pHs and high salinity. As other living cells, they use chemiosmotic mechanisms along with substrate level phosphorylation to conserve energy in form of ATP. Because some archaea are rooted close to the origin in the tree of life, these unusual mechanisms are considered to have developed very early in the history of life and, therefore, may represent first energy-conserving mechanisms. A key component in cellular bioenergetics is the ATP synthase. The enzyme from archaea represents a new class of ATPases, the A1A0 ATP synthases. They are composed of two domains that function as a pair of rotary motors connected by a central and peripheral stalk(s). The structure of the chemically-driven motor (A1) was solved by small-angle X-ray scattering in solution, and the structure of the first A1A0 ATP synthases was obtained recently by single particle analyses. These studies revealed novel structural features such as a second peripheral stalk and a collar-like structure. In addition, the membrane-embedded electrically-driven motor (A0) is very different in archaea with sometimes novel, exceptional subunit composition and coupling stoichiometries that may reflect the differences in energy-conserving mechanisms as well as adaptation to temperatures at or above 100 degrees C.

Overproduction of a functional A1 ATPase from the archaeon Methanosarcina mazei Gö1 in Escherichia coli.

Single subunits of the A1 ATPase from the archaeon Methanosarcina mazei Gö1 were produced in E. coli as MalE fusions and purified, and polyclonal antibodies were raised against the fusion proteins. A DNA fragment containing the genes ahaE, ahaC, ahaF, ahaA, ahaB, ahaD, and ahaG, encoding the hydrophilic A1 domain and part of the stalk of the A1AO ATPase of M. mazei Gö1, was constructed, cloned into an expression vector and transformed into different strains of Escherichia coli. In any case, a functional, ATP-hydrolysing A1 ATPase was produced. Western blots demonstrated the production of subunits A, B, C, and F in E. coli, and minicell analyses suggested that subunits D, E, and G were produced as well. This is the first demonstration of a heterologous production of a functional ATPase from an archaeon. The A1 ATPase was sensitive to freezing but lost only about 50% of its activity within 18 days on ice. Inhibitor studies revealed that the heterologously produced A1 ATPase is insensitive to azide, dicyclohexylcarbodiimide and bafilomycin A1, but sensitive to diethylstilbestrol and its analogues dienestrol and hexestrol. The expression system described here will open new avenues towards the functional and structural analyses of this unique class of enzymes.

Structural Insights into the A1 ATPase from the archaeon, Methanosarcina mazei Gö1.

The low-resolution structure and overall dimensions of the A(3)B(3)CDF complex of the A(1) ATPase from Methanosarcina mazei Gö1 in solution is analyzed by synchrotron X-ray small-angle scattering. The radius of gyration and the maximum size of the complex are 5.03 +/- 0.1 and 18.0 +/- 0.1 nm, respectively. The low-resolution shape of the protein determined by two independent ab initio approaches has a knob-and-stalk-like feature. Its headpiece is approximately 9.4 nm long and 9.2 nm wide. The stalk, which is known to connect the headpiece to its membrane-bound A(O) part, is approximately 8.4 nm long. Limited tryptic digestion of the A(3)B(3)CDF complex was used to probe the topology of the smaller subunits (C-F). Trypsin was found to cleave subunit C most rapidly at three sites, Lys(20), Lys(21), and Arg(209), followed by subunit F. In the A(3)B(3)CDF complex, subunit D remained protected from proteolysis.

Structure and function of the A1A0-ATPases from methanogenic Archaea.

Recent molecular studies revealed nine to ten gene products involved in function/assembly of the methanoarchaeal ATPase and unravel a close relationship of the A1A0-ATPase and the V1V0-ATPase with respect to subunit composition and the structure of individual subunits. Most interestingly, there is an astonishing variability in the size of the proteolipids in methanoarchaeal A1A0-ATPases with six, four, or two transmembrane helices and a variable number of conserved protonizable groups per monomer. Despite the structural similarities the A1A0-ATPase differs fundamentally from the V1V0-ATPase by its ability to synthesize ATP, a feature shared with F1F0-ATPases. The discovery of duplicated and triplicated versions of the proteolipid in A1A0-ATP synthases questions older views of the structural requirements for ATP synthases versus ATP hydrolases and sheds new light on the evolution of these secondary energy converters.

The A1A0 ATPase from Methanosarcina mazei: cloning of the 5' end of the aha operon encoding the membrane domain and expression of the proteolipid in a membrane-bound form in Escherichia coli.

Three additional ATPase genes, clustered in the order ahaH, ahaI, and ahaK, were found upstream of the previously characterized genes ahaECFABDG coding for the archaeal A1A0 ATPase from Methanosarcina mazei. ahaH, the first gene in the cluster, is preceded by a conserved promoter sequence. Northern blot analysis revealed that the clusters ahaHIK and ahaECFABDG are transcribed as one message. AhaH is a hydrophilic polypeptide and is similar to peptides of previously unassigned function encoded by genes preceding postulated ATPase genes in Methanobacterium thermoautotrophicum and Methanococcus jannaschii. AhaI has a two-domain structure with a hydrophilic domain of 39 kDa and a hydrophobic domain with seven predicted transmembrane alpha helices. It is similar to the 100-kDa polypeptide of V1V0 ATPases and is therefore suggested to participate in proton transport. AhaK is a hydrophobic polypeptide with two predicted transmembrane alpha helices and, on the basis of sequence comparisons and immunological studies, is identified as the proteolipid, a polypeptide which is essential for proton translocation. However, it is only one-half and one-third the size of the proteolipids from M. thermoautotrophicum and M. jannaschii, respectively. ahaK is expressed in Escherichia coli, and it is incorporated into the cytoplasmic membrane despite the different chemical natures of lipids from archaea and bacteria. This is the first report on the expression and incorporation into E. coli lipids of a membrane integral enzyme from a methanogens, which will facilitate analysis of the structure and function of the membrane domain of the methanoarchaeal ATPase.