Characterization of the Functionally Critical AXAXAXA and PXXEXXP Motifs of the ATP Synthase c-Subunit from an Alkaliphilic Bacillus.
The membrane-embedded rotor in the F(0) sector of proton-translocating ATP synthases is formed from hairpin-like c-subunits that are protonated and deprotonated during energization of ATP synthesis. This study focuses on two c-subunit motifs that are unique to synthases of extremely alkaliphilic Bacillus species. One motif is the AXAXAXA sequence found in the N-terminal helix-1 instead of the GXGXGXG of non-alkaliphiles. Quadruple A-->G chromosomal mutants of alkaliphilic Bacillus pseudofirmus OF4 retain 50% of the wild-type hydrolytic activity (ATPase) but <18% of the ATP synthase capacity at high pH. Consistent with a structural impact of the four alanine replacements, the mutant ATPase activity showed enhanced inhibition by dicyclohexylcarbodiimide, which blocks the helix-2 carboxylate. Single, double, or triple A-->G mutants exhibited more modest defects, as monitored by malate growth. The key carboxylate is in the second motif, which is P(51)XXE(54)XXP in extreme alkaliphiles instead of the (A/G)XX(E/D)XXP found elsewhere. Mutation of Pro(51) to alanine had been shown to severely reduce malate growth and ATP synthesis at high pH. Here, two Pro(51) to glycine mutants of different severities retained ATP synthase capacity but exhibited growth deficits and proton leakiness. A Glu(54) to Asp(54) change increased proton leakiness and reduced malate growth 79-90%. The Pro(51) and the Glu(54) mutants were both more dicyclohexylcarbodiimide-sensitive than wild type. The results highlight the requirement for c-subunit adaptations to achieve alkaliphile ATP synthesis with minimal cytoplasmic proton loss and suggest partial suppression of some mutations by changes outside the atp operon.
Liu
Jun
J
Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York 10029, USA.
Fujisawa
Makoto
M
Hicks
David B
DB
Krulwich
Terry A
TA
D000222:Adaptation, Physiological; D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D025243:Bacterial Proton-Translocating ATPases; D020134:Catalytic Domain / Q000502:physiology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D009154:Mutation; D016366:Open Reading Frames / Q000502:physiology; D009876:Operon / Q000502:physiology
D001426:Bacterial Proteins; D004024:Dicyclohexylcarbodiimide; D025243:Bacterial Proton-Translocating ATPases