Cystathionine β-synthase (CBS) domains discovered 20 years ago can bind different adenosine derivatives (AMP, ADP, ATP, S-adenosylmethionine, NAD, diadenosine polyphosphates) and thus regulate the activities of numerous proteins. Mutations in CBS domains of enzymes and membrane transporters are associated with several hereditary diseases. The regulatory unit is a quartet of CBS domains that belong to one or two polypeptides and usually form a conserved disk-like structure. CBS domains function as “internal inhibitors” in enzymes, and their bound ligands either amplify or attenuate the inhibitory effect. Recent studies have opened a way to understanding the structural basis of enzyme regulation via CBS domains and widened the list of their bound ligands.

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 P¹,Pⁿ-diadenosine 5′-polyphosphates (Ap_nAs, 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 Ap_nAs 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 Ap_nAs and showed no enthalpy change, indicating the importance of the DTRGG domain for Ap_nA binding. These findings suggest that Ap_nAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.

Ceruloplasmin (Cp) is a copper-containing multifunctional oxidase of plasma. New rat Cp features were discovered by X-ray analysis.

Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine β-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta. Enzyme interaction with substrate as characterized by the Michaelis constant (K_m) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg²⁺ except for the C. novyi PPase where Mg²⁺ produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller K_m value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PP_i level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.