An asparagine residue mediates intramolecular communication in nucleotide-regulated pyrophosphatase

Abstract

Many prokaryotic soluble PPases (pyrophosphatases) contain a pair of regulatory adenine nucleotide-binding CBS (cystathionine β-synthase) domains that act as 'internal inhibitors' whose effect is modulated by nucleotide binding. Although such regulatory domains are found in important enzymes and transporters, the underlying regulatory mechanism has only begun to come into focus. We reported previously that CBS domains bind nucleotides co-operatively and induce positive kinetic co-operativity (non-Michaelian behaviour) in CBS-PPases (CBS domain-containing PPases). In the present study, we demonstrate that a homodimeric ehPPase (Ethanoligenens harbinense PPase) containing an inherent mutation in an otherwise conserved asparagine residue in a loop near the active site exhibits non-co-operative hydrolysis kinetics. A similar N312S substitution in 'co-operative' dhPPase (Desulfitobacterium hafniense PPase) abolished kinetic co-operativity while causing only minor effects on nucleotide-binding affinity and co-operativity. However, the substitution reversed the effect of diadenosine tetraphosphate, abolishing kinetic co-operativity in wild-type dhPPase, but restoring it in the variant dhPPase. A reverse serine-to-asparagine replacement restored kinetic co-operativity in ehPPase. Molecular dynamics simulations revealed that the asparagine substitution resulted in a change in the hydrogen-bonding pattern around the asparagine residue and the subunit interface, allowing greater flexibility at the subunit interface without a marked effect on the overall structure. These findings identify this asparagine residue as lying at the 'crossroads' of information paths connecting catalytic and regulatory domains within a subunit and catalytic sites between subunits.

Tetrameric Structures of Inorganic CBS-Pyrophosphatases from Various Bacterial Species Revealed by Small-Angle X-ray Scattering in Solution

“…The primary sequence affects not only the overall shapes of the proteins, but also their functional properties. It has been shown earlier that Asn312 of the DHH domain is involved in kinetic cooperativity [40]. Its replacement by serine in dh-PPase led to the elimination of kinetic co-operativity in the enzyme and to the lack of kinetic cooperativity in eh-PPase, which contains a similar inherent mutation.…”

Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides

“…This helix interacts with the loops formed by residues 310–323 and 334–341; the former loop contains Asn312, earlier shown to be important for catalysis and kinetic cooperativity. 17 Next to the other side of helix α1 is Arg276, also important for allosteric regulation. 18…”

“…17,18 Asparagine substitution by a serine residue dramatically affects regulation by abolishing kinetic cooperativity in the absence of adenine nucleotides but restoring it in the presence of Ap 4 A. 17 The arginine located in the CBS2 domain has been found to control kinetic cooperativity, nucleotide-binding affinity, and the size of the regulatory signal exerted by both mono- and dinucleotides. 18 However, transmission of the regulatory signal from the CBS domains to a distantly located catalytic site and between subunits in CBS-PPase should involve an extended residue network that links all catalytic and regulatory sites in CBS-PPase.…”

The Tetrameric Structure of Nucleotide-regulated Pyrophosphatase and Its Modulation by Deletion Mutagenesis and Ligand Binding

“…D. hafniense CBS-PPase (dhPPase), its separate catalytic part (dhPPaseΔCDC), and the CBS-PPases of Clostridium perfringens (cpPPase), Clostridium novyi (cnPPase), Eggerthella lenta CBS-PPase (elPPase), and Ethanoligenens harbinense CBS-PPase (ehPPase) were produced and purified as described elsewhere [12,13,15]. The last two enzymes (elPPase and ehPPase) lack the DRTGG domain in their structures.…”