The ATP Synthase a-subunit of Extreme Alkaliphiles Is a
Makoto Fujisawa1, Oliver J. Fackelmayer, Jun Liu, Terry A. Krulwich, and David B. Hicks2
A lysine residue in the putative proton uptake pathway of the
ATP synthase a-subunit is found only in alkaliphilic Bacillus
and passage to the synthase rotor. Here, Lys-180 was replaced
with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg),
or histidine (His) in the chromosome of alkaliphilic Bacillus
pseudofirmus OF4. All mutants exhibited octylglucoside-
stimulated ATPase activity and ?-subunit levels at least as
high as wild-type. Purified mutant F1F0-ATP synthases all
contained substantial a-subunit levels. The mutants exhib-
ited diverse patterns of native (no octylglucoside) ATPase
activity and a range of defects in malate growth and in vitro
ATP synthesis at pH 10.5. ATP synthesis by the Ala, Gly, and
His mutants was also impaired at pH 7.5 in the presence of a
pH. The Arg mutant exhibited no ATP synthesis activity in
the alkaliphile setting although activity was reported for a
K180R mutant of a thermoalkaliphile synthase (McMillan,
D. G., Keis, S., Dimroth, P., and Cook, G. M. (2007) J. Biol.
Chem. 282, 17395–17404). The hypothesis that a-subunits
from extreme alkaliphiles and the thermoalkaliphile repre-
sent distinct variants was supported by demonstration of the
importance of additional alkaliphile-specific a-subunit resi-
dues, not found in the thermoalkaliphile, for malate growth
of B. pseudofirmus OF4. Finally, a mutant B. pseudofirmus
Gly-212 (helix 5) retained significant coupled synthase activ-
ity accompanied by proton leakiness.
Proton-coupled F1F0-ATP synthases are centrally important
for non-fermentative cells that energize ATP synthesis using
the energy of an electrochemical proton gradient, the PMF,3
across the cytoplasmic or thylakoid membrane (bacteria) and
across the mitochondrial or chloroplast thylakoid membrane
(eukaryotes) (1–3). ATP synthases are composed of two do-
mains, with bacterial synthases having simpler structures than
single ?-, ?-, and ?-subunits. The membrane-associated F0
domain is composed of a single a-subunit, two b-subunits, and
multiple c-subunits (2, 4–6). ATP synthases function as rotary
nano-machines, in which inward translocation of protons
through the F0domain leads to rotation of a membrane-em-
bedded ring-like rotor (2, 3, 7–10). The rotor is formed from
10–15 hairpin-like c-subunits, depending upon the organism
(11–16). Essential steps in coupling of ATP synthesis to the
PMF include the protonation of successive c-subunits of the
rotor and, after full rotation of a protonated c-subunit, de-pro-
tonation of that subunit through interactions of c-subunits of
extensive biochemical and genetic evidence indicates that this
ATP synthase subunit plays roles in providing the proton path
from outside the membrane surface to the carboxylates of
served arginine in TMH4 (Arg-210 in Escherichia coli) is pro-
rotation (25) and to cause a shift in the pKaof the essential
carboxylate so that the proton that has completed rotation dis-
plasm. That proton exit pathway is also likely to be within the
a-subunit (4, 5, 18, 23, 26–28).
Valuable insights into the mechanism of ATP synthase have
29–31). Our own studies have focused on the ATP synthase of
alkaliphilic Bacillus species. The model extreme alkaliphile for
bioenergetic studies is Bacillus pseudofirmus OF4, which is
using proton-coupled oxidative phosphorylation, at external
pH values from 7.5 to ?11 (30, 32–34). B. pseudofirmus OF4
and other alkaliphilic Bacillus species share sequence motifs in
the a- and c-subunits of their proton-coupled ATP synthases
(30, 33, 35). Initial mutagenesis work showed that several of
these sequence deviations from the neutralophilic Bacillus
consensus have indispensible roles in the synthetic function
of the enzyme and non-fermentative growth on malate at
high pH, but are not required for hydrolytic ATPase activity
Grant GM28454 from NIGMS.
1Current address: Faculty of Food Life Sciences, Toyo University, 1-1-1
Izumino, Itakura machi, Ora-gun, Gunma 374-0193, Japan.
2To whom correspondence should be addressed: Dept. of Pharmacology &
Systems Therapeutics, Mount Sinai School of Medicine, One Gustave L.
Levy Place, New York, New York 10029. Tel.: 212-241-7466; Fax: 212-996-
7214; E-mail: firstname.lastname@example.org.
CCCP, carbonyl m-chlorophenylhydrazone; DCCD, N,N?-dicyclohexylcar-
bodiimide; OG, octylglucoside; RSO, right-side out; TMH, transmembrane
helix; MOPS, 4-morpholinepropanesulfonic acid; NTA, nitrilotriacetic acid.
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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
OCTOBER 15, 2010•VOLUME 285•NUMBER 42JOURNAL OF BIOLOGICAL CHEMISTRY 32105
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or non-fermentative growth of B. pseudofirmus OF4 near
neutral pH (36, 37). Recent structural studies on the B.
pseudofirmus OF4 tri-decameric c-rotor have begun to pro-
vide insights into how the major motifs of the alkaliphile
c-subunits impact the structure and support the function of
the rotor at high pH (31).
The current study focuses attention on alkaliphile-specific
conflicting observations on a particular “alkaliphile-specific”
residue, Lys-180, that is found both in extremely alkaliphilic
Bacillus species, e.g. B. pseudofirmus OF4, Bacillus halodurans
C-125, and Bacillus alcalophilus, and in more temperate alka-
liphiles such as Bacillus clausii and thermoalkaliphilic Caldal-
kalibacillus thermarum TA2.A1 (formerly called Bacillus sp.
TA2.A1). This residue is located in the TMH4 in the putative
proton uptake pathway of the a-subunit. TMH4 also contains
the conserved, functionally critical arginine (Arg-172 in B.
pseudofirmus OF4) in the proton pathway through the a-sub-
unit (17, 18, 22). The unusual Lys-180 substitutes for a glycine
residue typically found at this position in non-alkaliphiles (Fig.
1A). Mutagenesis studies of Lys-180 of B. pseudofirmus OF4
showed that its mutation to the consensus glycine of non-alka-
liphiles resulted in a major deficit in ATP synthesis at pH 10.5,
while significant capacity for ATP synthesis was retained at
near neutral pH (37). We hypothesized that, at high pH, Lys-
180 is a participant of the proton uptake path that is required
for capture of entering protons and passage of the protons to
the interface of the c-ring with the a-subunit. Subsequently,
operon from C. thermarum TA2.A1 in an E. coli mutant from
which the native atp operon was deleted. They found that this
thermoalkaliphile enzyme failed to support non-fermentative
8.0. Robust support of ATP synthesis at pH 7.0–7.5 was con-
ferred upon the enzyme when histidine or glycine replaced the
lysine, whereas replacement of Lys-180 with arginine resulted
a synthase with Lys-180 might be unable to synthesize ATP at
near neutral or neutral pH (38). The subsequent finding that C.
thermarum TA2.A1 could not grow non-fermentatively below
pH 8.0 was consistent with that proposal (39). However, the
proposal was at odds with our observation of robust non-fer-
mentative growth and ATP synthesis at pH 7.5 exhibited by
wild-type B. pseudofirmus OF4, which utilizes a Lys-180-con-
a-subunits from diverse mesophilic Bacillus species and ther-
moalkaliphilic C. thermarum TA2.A1 revealed that there were
many instances in which the thermoalkaliphile sequence de-
parted from the consensus sequence for extremely alkaliphilic
or from all mesophilic Bacillus strains, whether alkaliphilic or
neutralophilic. Moreover, those residues were in functionally
the a-subunits from thermoalkaliphile and extreme alkali-
philes, which are not also thermophiles, are distinct variants.
Presumably, the thermoalkaliphile variant reflects adaptations
the extreme alkaliphile variant reflects adaptations that under-
pin function in a more highly alkaline range. If so, it was likely
that, if the Lys-180 of B. pseudofirmus OF4 was replaced with
the same residues that had been introduced into the thermoal-
kaliphile synthase, their effects would be very different because
of the distinct scaffold into which they were introduced. Here,
mus OF4 that were made in the native atp locus of the alkali-
thermarum TA2.A1 synthase (38). In addition, we constructed
FIGURE 1. Alignment of the a-subunit putative transmembrane helices 4 and 5 and a diagrammatic representation of all the mutants in the study.
alkaliphiles B. pseudofirmus OF4 (AAG48358), B. halodurans C-125 (NP_244625.1) and B. alcalophilus (P25965), the more moderately alkaliphilic B. clausii
licheniformis (YP_093439), B. megaterium (AAA82520), B. subtilis 168 (NP_391568), and G. kaustophilus HTA42 (YP_149217); also included is the model Gram-
although not in B. clausii or B. selenitireducens MLS10. Two additional deviations from the consensus sequence for non-alkaliphilic Bacillus species that are
found in all of the alkaliphiles except for thermoalkaliphile C. thermarum TA2.A1 are boxed for alkaliphiles (Met-171 and Met-184) and underlined for the
thermoalkaliphile. Some residues only found in most alkaliphiles but not all alkaliphiles are also boxed (Val-177, Ile-185, and Leu-205). B, a topological
representation of the putative five-transmembrane helix structure of the B. pseudofirmus OF4 a-subunit with the location of the residues in TMH4 and -5 that
were mutated as well as selected alkaliphile-specific residues of interest in other regions that are not conserved in the a-subunit from thermoalkaliphile C.
thermarum; the replacements made in each location are indicated. Residues mutated to the residue found in C. thermarum at that position have a white
background while other mutations have a black background.
32106 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 285•NUMBER 42•OCTOBER 15, 2010
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Liu, Terry A. Krulwich and David B. Hicks
2010, 285:32105-32115. J. Biol. Chem.
Makoto Fujisawa, Oliver J. Fackelmayer, Jun
SUPPORT ALKALIPHILE OXIDATIVE
LYS-180 AND OTHER RESIDUES THAT
MUTATIONS IN THE CRITICAL
Alkaliphiles Is a Distinct Variant:
The ATP Synthase a-subunit of Extreme
doi: 10.1074/jbc.M110.165084 originally published online August 17, 2010
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