A CBS domain-containing pyrophosphatase ofMoorella thermoaceticais regulated by adenine nucleotides

Abstract

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.

Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria

“…In this respect, Ap n As partially substitute for Mg 2ϩ as an enzyme activator. Qualitatively similar activating effects on CBS-PPases were previously observed with ATP (31,32), although ATP effects were smaller in size and required much higher effector concentrations. A further difference is that ATP bound cooperatively, like Ap 3 A.…”

“…Interestingly, only in CBS-PPases (but not all of them), are the CBS domains intercalated by another (DRTGG) domain. CBS-PPases are activated by ATP and inhibited by AMP and ADP (31,32). Both catalysis and regulation involve marked positive cooperativity, which is Mg 2ϩ -dependent (32).…”

Cystathionine β-Synthase (CBS) Domains Confer Multiple Forms of Mg2+-dependent Cooperativity to Family II Pyrophosphatases

“…Micromolar concentrations of AMP and ADP caused inhibition in most cases, whereas ATP acted as an activator. Qualitatively similar effects of these nucleotides were reported previously for the CBS domain-containing mtPPase (18). The major new finding here is that, in most cases where the size of the effect allowed quantitative analysis, the curves were poorly described in terms of a simple 1:1 binding model (Equation 3) but obeyed Equation 2 for cooperative binding to two sites with concomitant activation or inhibition.…”

“…We reported previously that mtPPase (18) and the CBS domain-containing PPase of Clostridium perfringens (cpPPase) (19) are highly sensitive to micromolar concentrations of adenine nucleotides. Remarkably, AMP and ADP acted as inhibitors, whereas ATP and AP 4 A were activators.…”

IMP Dehydrogenase Type 1 Associates with Polyribosomes Translating Rhodopsin mRNA

“…The enzyme is a homotetramer; each monomer is composed of a catalytic (␤/␣) 8 barrel and a subdomain containing two CBS domains (named for the related domain in cystathionine ␤-synthase) (Fig. 1).…”