title: Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria.
abstract: Among numerous proteins containing pairs of regulatory cystathionine β-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P(1),P(n)-diadenosine 5'-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ~10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.
authors.affiliation: From the Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and the Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia.
authors.forename: Viktor A
authors.initials: VA
authors.lastname: Anashkin
authors.affiliation: From the Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and.
authors.forename: Anu
authors.initials: A
authors.lastname: Salminen
authors.affiliation: From the Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and.
authors.forename: Heidi K
authors.initials: HK
authors.lastname: Tuominen
authors.affiliation: the Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia.
authors.forename: Victor N
authors.initials: VN
authors.lastname: Orlov
authors.affiliation: From the Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and reijo.lahti@utu.fi.
authors.forename: Reijo
authors.initials: R
authors.lastname: Lahti
authors.affiliation: the Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia baykov@genebee.msu.su.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Baykov
mesh_terms: D000227:Adenine Nucleotides / Q000737:chemistry; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D003016:Clostridium perfringens / Q000201:enzymology; D003541:Cystathionine beta-Synthase / Q000737:chemistry*; D015226:Dinucleoside Phosphates / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D058845:Moorella / Q000201:enzymology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry*
chemical_list: D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D015226:Dinucleoside Phosphates; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; C020733:diadenosine tetraphosphate; C011440:diadenosine triphosphate; C070070:diadenosine 5',5''''-P1,P6-hexaphosphate; D011755:Pyrophosphatases; D003541:Cystathionine beta-Synthase
keywords: CBS domain; allosteric regulation; bacterial signal transduction; calorimetry; cooperativity; diadenosine polyphosphate; enzyme kinetics; inorganic pyrophosphatase; stress response