The membrane-associated ATPase from Sulfolobus acidocaldarius is distantly related to F1-ATPase as assessed from the primary structure of its alpha-subunit

Isolation of novel membrane-associated ATPases, presumably soluble parts of the H+-ATPases, from archaebacteria has been recently reported, and their properties were found to be significantly different from the usual F1-ATPase. In order to assess the relationship of the archaebacterial ATPases to the F1-ATPases and other known ATPases, the amino acid sequence of the alpha subunit of the ATPase from Sulfolobus acidocaldarius, an acidothermophilic archaebacterium, was compared with the sequences of other ATPases. The gene encoding its alpha subunit was cloned from the genomic library of S. acidocaldarius, and the nucleotide sequence was determined. The 591-amino acid sequence deduced from the nucleotide sequence contains a small number of short stretches that shows sequence similarity to the alpha and beta subunits of F1-ATPase. However, the overall similarity is too weak to consider it to be a typical member of the F1-ATPase family when the highly conserved sequences of the F1-ATPase subunits among various organisms are taken into account. Moreover, most of these stretches overlap the consensus sequences that are commonly found in some nucleotide-binding proteins. There is no significant sequence similarity to the ion-translocating ATPases, which form phosphorylated intermediates, such as animal Na+,K+-ATPases. Thus, the S. acidocaldarius ATPase and probably other archaebacterial ATPases also appear to belong to a new group of ion-translocating ATPases that has only a distant relationship to F1-ATPase.     

Molecular cloning of the beta-subunit of a possible non-F0F1 type ATP synthase from the acidothermophilic archaebacterium, Sulfolobus acidocaldarius

The gene which encodes the beta subunit of the novel membrane-associated ATPase has been identified and characterized. The beta subunit, which is most likely the soluble part of the non-F0F1 type H+-ATPase, was obtained from the archaebacterium, Sulfolobus acidocaldarius. In terms of its location, it follows just after the gene for its alpha subunit. It is comprised of 1398 nucleotides, corresponding to a protein of 465 amino acids, and the consensus sequence in the nucleotide binding proteins is poorly conserved. Together with previously described results, the distant homology of the S. acidocaldarius ATPase alpha and beta subunits when compared to those of F0F1-ATPases indicates that this archaebacterial ATPase belongs to an ion-translocating ATPase family uniquely different than F0F1-ATPases even if S. acidocaldarius ATPase and F0F1-ATPases have been derived from a common ancestral ATPase.     

A gene encoding the proteolipid subunit of Sulfolobus acidocaldarius ATPase complex

An analysis of genes for the major two subunits of the membrane-associated ATPase from an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, suggested that it belongs to a different ATPase family from the F1-ATPase (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1988) J. Biol. Chem. 263, 17251-17254). In the same operon of the above two genes we found a gene encoding a very hydrophobic protein of 101 amino acids (Mr = 10,362). A proteolipid was purified from the membranes of this bacteria in which partial amino acid sequences matched with the sequence deduced from the gene. Significant amino acid sequence homology and a similar hydropathy profile appeared when the sequence was compared with the 8-kDa proteolipid subunit of F0F1-ATPases. It is about 30 amino acids larger than the 8-kDa proteolipid and has a small (11-amino acid) repeat sequence. However, it is distinct from the 16-kDa proteolipid subunit of an eukaryotic vacuolar H+-ATPase (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,5521-5524).     

Correspondence of minor subunits of plant mitochondrial F1ATPase to F1F0ATPase subunits of other organisms

In addition to two major alpha- and beta-subunits, the soluble oligomycin-insensitive F1ATPase purified from sweet potato root mitochondria contains four different minor subunits of gamma (Mr = 35,500), delta (Mr = 27,000), delta' (Mr = 23,000), and epsilon (Mr = 12,000) (Iwasaki, Y., and Asashi, T. (1983) Arch. Biochem. Biophys. 227, 164-173). Among these minor subunits, the delta-subunit specifically cross-reacted with an antibody against the delta-subunit of maize mitochondrial F1 which contains only three minor gamma-, delta- and epsilon-subunits like F1ATPases from other organisms, indicating that the delta'-subunit is an extra subunit of sweet potato F1 which is absent in the maize F1. All of the four minor subunits of sweet potato F1 were purified and their N-terminal amino acid sequences of 30-36 residues were determined. The N-terminal sequence of gamma-subunit was homologous to those of the gamma-subunits of bacterial F1 and mammalian mitochondrial F1. The N-terminal sequence of the delta-subunit was homologous to those of the delta-subunits of bacterial F1, chloroplast CF1, and oligomycin sensitivity conferring protein of bovine mitochondrial F1F0. A sequence homology was also observed between the sweet potato epsilon-subunit and the epsilon-subunit of bovine mitochondrial F1. The N-terminal sequence of the delta'-subunit did not show any significant sequence homology to known protein sequences. These subunit correspondences place plant mitochondrial F1 at an unique position in the evolution of F1ATPase.     

The cDNA sequence of the 69-kDa subunit of the carrot vacuolar H+-ATPase. Homology to the beta-chain of F0F1-ATPases

Vacuolar ATPases constitute a novel class of N-ethylmaleimide- and nitrate-sensitive proton pumps associated with the endomembrane system of eukaryotic cells. They resemble F0F1-ATPases in that they are large multimeric proteins, 400-500 kDa, composed of three to nine different subunits. Previous studies have indicated that the active site is located on the approximately 70-kDa subunit. Using antibodies to the approximately 70-kDa subunit of corn to screen a carrot root lambda gt11 cDNA library, we have isolated cDNA clones of the carrot 69-kDa subunit. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA. The 69-kDa subunit consists of 623 amino acids (Mr 68,835), with no obvious membrane-spanning regions. The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone. The protein sequence of the carrot 69-kDa subunit also exhibited 34.3% identity to four representative F0F1-ATPase beta-chains over a 275-amino-acid core stretch of similar sequence. Alignment studies revealed several regions which were highly homologous to beta-chains, including sequences previously implicated in catalytic function. This provides definitive evidence that the vacuolar ATPase is closely related to the F0F1-type ATPases. A major functional difference between the 69-kDa and beta-subunits is the location of 3 critical cysteine residues: two in the putative catalytic region (Cys-248 and Cys-256) and one in the proposed Mg2+-binding site (Cys-279). These cysteines (and two others) probably account for the sensitivity of the vacuolar H+-ATPase to the sulfhydryl reagent, N-ethylmaleimide. It is proposed that the two ATPases may have arisen from a common ancestor by the insertion or deletion of a large stretch of nonhomologous sequence near the amino-terminal end of the subunit.     

A DNA fragment homologous to F1-ATPase beta subunit was amplified from genomic DNA of Methanosarcina barkeri. Indication of an archaebacterial F-type ATPase.

A 490 bp DNA fragment was amplified from Methanosarcina barkeri genomic DNA by the polymerase chain reaction (PCR) using oligonucleotide primers designed based on conserved amino acid sequences of the F1-ATPase beta subunits. The amino acid sequence deduced from the DNA sequence of this fragment was highly homologous to a portion of the F1-ATPase beta subunit. This indicates that this archaebacterium has a gene of F-type ATPase in addition to a gene of V-type ATPase.     

Redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch

F(1)-ATP synthase (F(1)-ATPase) is equipped with a special mechanism that prevents the wasteful reverse reaction, ATP hydrolysis, when there is insufficient proton motive force to drive ATP synthesis. Chloroplast F(1)-ATPase is subject to redox regulation, whereby ATP hydrolysis activity is regulated by formation and reduction of the disulfide bond located on the γ subunit. To understand the molecular mechanism of this redox regulation, we constructed a chimeric F(1) complex (α(3)β(3)γ(redox)) using cyanobacterial F(1), which mimics the regulatory properties of the chloroplast F(1)-ATPase, allowing the study of its regulation at the single molecule level. The redox state of the γ subunit did not affect the ATP binding rate to the catalytic site(s) and the torque for rotation. However, the long pauses caused by ADP inhibition were frequently observed in the oxidized state. In addition, the duration of continuous rotation was relatively shorter in the oxidized α(3)β(3)γ(redox) complex. These findings lead us to conclude that redox regulation of CF(1)-ATPase is achieved by controlling the probability of ADP inhibition via the γ subunit inserted region, a sequence feature observed in both cyanobacterial and chloroplast ATPase γ subunits, which is important for ADP inhibition (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865).     

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.     

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Replacement of serine 373 by phenylalanine in the alpha subunit of Escherichia coli F1-ATPase results in loss of steady-state catalysis by the enzyme

The mutant allele (uncA401) of the gene for the alpha subunit of Escherichia coli F1-ATPase was cloned from the total DNA of the mutant AN120 on a hybrid plasmid pAN120. Determination of the DNA sequence of the alpha subunit gene from pAN120 revealed a single base change of cytosine at nucleotide residue 1118 to thymine and indicated that serine 373 was replaced by phenylalanine. It has been reported that the mutant F1 is defective in a step of steady-state catalysis, whereas its single turnover process is normal (Kanazawa, H., Noumi, T., Matsuoka, I., Hirata T., and Futai, M. (1984) Arch. Biochem. Biophys. 228, 258-269). Thus, we concluded that serine 373 in the alpha subunit is essential for steady-state catalysis by F1-ATPase.     

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.     

一种免疫生物传感器对单核细胞增生李斯特菌快速检测方法的初步研究

应用F0F1-ATP酶旋转分子马达和免疫技术相结合,建立免疫生物传感器快速检测技术。首先pH变化敏感荧光物质F1300标记到色素体(chrom atophore)的内表面,然后在F0F1-ATP酶上连接β亚基抗体-生物素-链亲和素-生物素-单核细胞增生李斯特菌多抗复合体,得到可以捕获单核细胞增生李斯特菌的免疫生物传感器。传感器上负载菌量不同,酶活性不同,酶活变化以pH敏感的荧光探针来感应,最后通过荧光扫描仪检测不同菌量负载下的荧光信号。结果表明,该方法对单核细胞增生李斯特菌标准菌株(ATCC 15313)的检测时间为4.5 h,检出浓度为100 CFU/孔。     

Formation of an energized inner membrane in mitochondria with a gamma-deficient F1-ATPase

Eukaryotic cells require mitochondrial compartments for viability. However, the budding yeast Saccharomyces cerevisiae is able to survive when mitochondrial DNA suffers substantial deletions or is completely absent, so long as a sufficient mitochondrial inner membrane potential is generated. In the absence of functional mitochondrial DNA, and consequently a functional electron transport chain and F(1)F(o)-ATPase, the essential electrical potential is maintained by the electrogenic exchange of ATP(4-) for ADP(3-) through the adenine nucleotide translocator. An essential aspect of this electrogenic process is the conversion of ATP(4-) to ADP(3-) in the mitochondrial matrix, and the nuclear-encoded subunits of F(1)-ATPase are hypothesized to be required for this process in vivo. Deletion of ATP3, the structural gene for the gamma subunit of the F(1)-ATPase, causes yeast to quantitatively lose mitochondrial DNA and grow extremely slowly, presumably by interfering with the generation of an energized inner membrane. A spontaneous suppressor of this slow-growth phenotype was found to convert a conserved glycine to serine in the beta subunit of F(1)-ATPase (atp2-227). This mutation allowed substantial ATP hydrolysis by the F(1)-ATPase even in the absence of the gamma subunit, enabling yeast to generate a twofold greater inner membrane potential in response to ATP compared to mitochondria isolated from yeast lacking the gamma subunit and containing wild-type beta subunits. Analysis of the suppressing mutation by blue native polyacrylamide gel electrophoresis also revealed that the alpha(3)beta(3) heterohexamer can form in the absence of the gamma subunit.     

The coupled chemomechanics of the F(1)-ATPase molecular motor

The enzyme F(1)-ATPase is a rotary nanomotor in which the central gamma subunit rotates inside the cavity made of alpha(3)beta(3) subunits. The experiments showed that the rotation proceeds in steps of 120 degrees and each 120 degrees step consists of 80 degrees and 40 degrees substeps. Here the Author proposes a stochastic wave mechanics of the F(1)-ATPase motor and combines it with the structure-based kinetics of the F(1)-ATPase to form a chemomechanic coupled model. The model can reproduce quantitatively and explain the experimental observations about the F(1) motor. Using the model, several rate-limited situations about gamma subunit rotation are proposed, the effects of the friction and the load on the substeps are investigated and the chemomechanic coupled time during ATP hydrolysis cycle is determined.     

cDNA sequence and homologies of the "57-kDa" nucleotide-binding subunit of the vacuolar ATPase from Arabidopsis

Functional and structural similarities among a wide variety of endomembrane H+-ATPases suggest that they form a distinct class with a common origin. Immunological studies (Manolson, M. F., Percy, J. M., Apps, D. K., Xie, X. S., Stone, D. K., and Poole, R. J. (1987) in Proceedings of the Membrane Protein Symposium (Goheen, S. C., ed) pp. 427-434, Bio-Rad, Richmond, CA, and M. F. Manolson, J. M. Percy, D. K. Apps, X. S. Xie, D. K. Stone, M. Harrison, D. J. Clarke, R. J. Poole, unpublished data) support this idea and suggest an evolutionary relationship between the endomembrane and F0F1 ATPases. Further examination of relationships necessitates comparison of protein/nucleic acid sequence data. To this end, we have cloned and sequenced the cDNA encoding the 57-kDa polypeptide of the Arabidopsis vacuolar membrane H+-ATPase. To our knowledge, this is the first report of the sequence of a "57-kDa" subunit for plant or animal endomembrane H+-ATPase. This cDNA encodes a hydrophilic polypeptide containing a putative ATP binding site. Lack of a secretion signal sequence suggests it is not processed through the endoplasmic reticulum but translated on cytosolic ribosomes. Comparison of protein sequences shows the 57-kDa subunit from Arabidopsis to be nearly identical with the corresponding subunit in Neurospora vacuolar membrane H+-ATPase, very similar to the beta subunit of the archaebacterium Sulfolobus, and slightly, but nevertheless significantly, homologous to the alpha and beta subunits of the F0F1-ATPases. These results suggest that these different classes of ATPases have evolved from a common ancestor.     

Molecular cloning of genes encoding major two subunits of a eubacterial V-type ATPase from Thermus thermophilus.

The atpAB genes which encode the alpha and beta subunits of membrane ATPase from a thermophilic eubacterium, Thermus thermophilus HB8, were cloned. The deduced amino-acid sequences of the alpha subunit (583 amino acids) and the beta subunit (478 amino acids) are only moderately similar to the alpha beta subunits of the F0F1-ATPases, while they are highly similar to the major two subunits of the V-type ATPases, a family of ATPases which have been so far found in eukaryotic endomembrane vacuolar vesicles and archaebacterial plasma membranes. Thus, T. thermophilus ATPase belongs to the V-type ATPase family, even though this bacterium is a eubacterium. The hypothesis that the differentiation of an ancestral ATPase into V-type and F0F1-ATPase occurred after the evolution of a primordial cell into archaebacteria and eubacteria should be modified accordingly.     

Coupling factor F1 ATPase with defective beta subunit from a mutant of Escherichia coli

The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form. The final specific activity of Mg2+-ATPase was 6-9 units/mg protein, which is about 10-15 times lower than that of F1 ATPase from the wild-type strain. The mutant F1 had a ratio of Ca2+-ATPase to Mg2+-ATPase of about 3.5, whereas the wild-type F1 had ratio of about 0.8. The mutant F1 was more unstable than wild-type F1: on storage at -80 degrees C for 2 weeks, about 80% of its activity (dependent on Ca2+ or Mg2+) was lost, whereas none of the activity of the wild-type F1 was lost. The following results indicate that the mutation is in the beta subunit. (i) High Mg2+-ATPase activity (about 20 units/mg protein) was reconstituted when the beta subunit from wild type F1 was added to dissociated mutant F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. (ii) Low ATPase activity having the same ratio of Ca2+-ATPase to Mg2+-ATPase as the mutant F1 was reconstituted when a mixture of the beta subunit from the mutant F1 and the alpha and gamma subunits from wild-type F1 was dialyzed against the same buffer. (iii) Tryptic peptide analysis of the beta subunit of the mutant showed a difference in a single peptide compared with the wild-type strain.     

Stable structure of thermophilic proton ATPase beta subunit

F1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes. F1-ATPase obtained from thermophilic bacterium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure. Its beta subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E. coli alpha and gamma subunits. Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the beta subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of beta's of three major categories of F1's; prokaryotic membranes, chloroplasts, and mitochondria. The following results were obtained. Homology: The primary structure of the TF1 beta subunit (473 residues, Mr = 51,995.6) showed 89.3% homology with 270 residues which are identical in the beta subunits from human mitochondria, spinach chloroplasts, and E. coli. It contained regions homologous to several nucleotide-binding proteins. Secondary structure: The deduced alpha-helical (30.1%) and beta-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra. Residues forming reverse turns (Gly and Pro) were highly conserved among the F1 beta subunits. Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1 beta subunit with those of the other F1 beta subunits mentioned above. The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure. Codon usage: The codon usage of the TF1 beta gene was found to be unique. The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cytosine, except in codons for Gln, Lys, and Glu.     

Nucleotide-binding sites in V-type Na+-ATPase from Enterococcus hirae

Enterococcus hirae V-ATPase, in contrast to most V-type ATPases, is resistant to N-ethylmaleimide (NEM). Alignment of the amino acid sequences of NtpA suggests that the NEM-sensitive Cys of V-type ATPases is replaced by Ala in E. hirae V-ATPase. Consistent with this prediction, the V-ATPase became sensitive upon substitution of the Ala with Cys. The three-dimensional structure of the NtpB subunit of V-ATPase was modeled based on the structure of the corresponding subunit (alpha subunit) of bovine F(1)-ATPase by homology modeling. Overall, the 3D structure of the subunit resembled that of alpha subunit of bovine F(1)-ATPase. The NtpB subunit, which lacks the P-loop consensus sequence for nucleotide binding, was predicted to bind a nucleotide at the modeled nucleotide-binding site. Experimental data supported the prediction that the E. hirae V-ATPase had about six nucleotide-binding sites.