A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides.
CBS (cystathionine beta-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family II PPase (inorganic pyrophosphatase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino-acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co2+ or Mn2+) for activity, it resembles common family II PPases, which lack CBS domains. The mtCBS-PPase, however, has lower activity than common family II PPases, is potently inhibited by ADP and AMP, and is activated up to 1.6-fold by ATP. Inhibition by AMP is competitive, whereas inhibition by ADP and activation by ATP are both of mixed types. The nucleotides are effective at nanomolar (ADP) or micromolar concentrations (AMP and ATP) and appear to compete for the same site on the enzyme. The nucleotide-binding affinities are thus 100-10000-fold higher than for other CBS-domain-containing proteins. Interestingly, genes encoding CBS-PPase occur most frequently in bacteria that have a membrane-bound H+-translocating PPase with a comparable PP(i)-hydrolysing activity. Our results suggest that soluble nucleotide-regulated PPases act as amplifiers of metabolism in bacteria by enhancing or suppressing ATP production and biosynthetic reactions at high and low [ATP]/([AMP]+[ADP]) ratios respectively.
Jämsen
Joonas
J
Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland.
Tuominen
Heidi
H
Salminen
Anu
A
Belogurov
Georgiy A
GA
Magretova
Natalia N
NN
Baykov
Alexander A
AA
Lahti
Reijo
R
D000227:Adenine Nucleotides / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D043564:Inorganic Pyrophosphatase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D046950:Thermoanaerobacterium / Q000201:enzymology*
D000227:Adenine Nucleotides; D043564:Inorganic Pyrophosphatase
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