An Unusual, His-dependent Family I Pyrophosphatase from Mycobacterium tuberculosis

Abstract

Soluble inorganic pyrophosphatases (PPases) comprise two evolutionarily unrelated families (I and II). These two families have different specificities for metal cofactors, which is thought to be because of the fact that family II PPases have three active site histidines, whereas family I PPases have none. Here, we report the structural and functional characterization of a unique family I PPase from Mycobacterium tuberculosis (mtPPase) that has two His residues (His 21 and His 86 ) in the active site. The 1.3-Å three-dimensional structure of mtPPase shows that His 86 directly interacts with bound sulfate, which mimics the product phosphate. Otherwise, mtPPase is structurally very similar to the well studied family I hexameric PPase from Escherichia coli, although mtPPase lacks the intersubunit metal binding site found in E. coli PPase. The cofactor specificity of mtPPase resembles that of E. coli PPase in that it has high activity in the presence of Mg 2؉ , but it differs from the E. coli enzyme and family II PPases because it has much lower activity in the presence of Mn 2؉ or Zn 2؉ . Replacements of His 21 and His 86 in mtPPase with the residues found in the corresponding positions of E. coli PPase had either no effect on the Mg 2؉ -and Mn 2؉ -supported reactions (H86K) or reduced Mg 2؉ -supported activity (H21K). However, both replacements markedly increased the Zn 2؉ -supported activity of mtPPase (up to 11-fold). In the double mutant, Zn 2؉ was a 2.5-fold better cofactor than Mg 2؉ . These results show that the His residues in mtPPase are not essential for catalysis, although they determine cofactor specificity. Soluble inorganic pyrophosphatase (PPase)3 (EC 3.6.1.1), is an essential metal-dependent enzyme that converts pyrophosphate into orthophosphate. This simple reaction provides a thermodynamic pull for many biosynthetic reactions that yield pyrophosphate as a byproduct (1). Soluble PPases belong to two nonhomologous families (2, 3): family I PPases, which are fairly widespread in all types of organisms, and family II PPases, which are exclusive to bacteria. In eubacteria and archaebacteria, the family I PPases are usually homohexamers, whereas in eukaryotes, they are homodimers. In contrast, all family II PPases are homodimers. The subunit size is generally 19 -22 kDa in hexameric PPases and 31-34 kDa in dimeric forms. Despite the variability of the subunit size in family I PPases, their active site cavities are formed by the same 13 functionally important polar residues, which is reflected in the conservation of the catalytic mechanism (4, 5). Although no sequence or overall structural similarity is observed between the two families, there is a striking similarity in the spatial arrangement of six key active site residues, a remarkable example of convergent enzyme evolution (6, 7).Functionally, family II differs from family I in its preference for Mn 2ϩ over Mg 2ϩ as a cofactor. Mg 2ϩ is a better cofactor for family I PPases, and it also activates family II PPases to the same extent. However, Mn 2ϩ co...

Structural and computational dissection of the catalytic mechanism of the inorganic pyrophosphatase from Mycobacterium tuberculosis

“…These conformational differences may result from evolutionary divergent sequences of Mtb and E. coli PPiases. For example, His21, one of the two His unique to Mtb (Tammenkoski et al, 2005), is wedged between Asp54 and Phe44, forming a salt bridge with Asp54, a hydrogen bond with the carbonyl oxygen of Val19 stacking perpendicularly with Phe44 and sterically interacting with Pro55. Thus, His21 appears to keep loop 1 away from the PP i .…”

“…Therefore, the order of product dissociation events in the catalytic turnover of bacterial PPiases remains to be clarified. In addition, the active site of Mtb PPiase displays unique features (Benini and Wilson, 2011;Tammenkoski et al, 2005), such as two His residues that are not present in E. coli and whose role in catalysis is not clear. These His residues appear to be too far to interact with the substrate directly, but they can help orient the residues that are directly involved in catalysis.…”

“…These His residues appear to be too far to interact with the substrate directly, but they can help orient the residues that are directly involved in catalysis. A double mutant of the His residues reduced k cat 5-fold, demonstrating a modest effect (Tammenkoski et al, 2005).…”

“…Because of the global clinical significance of Mtb as a deadly pathogen, Mtb PPiase is considered to be a potential target for novel anti-tuberculosis drugs (Biswas et al, 2013;Lv et al, 2014). In addition to extensive structural and biochemical studies on E. coli and S. cerevisiae PPiases Heikinheimo et al, 2000;Merckel et al, 2001;Pohjanjoki et al, 2001;Samygina et al, 2007), X-ray crystallographic studies were also carried out with the Mtb PPiase (Benini and Wilson, 2011;Tammenkoski et al, 2005), providing structural information on the location of a sole phosphate ion in the active site (PDB ID: 1WCF). The position of this P i , presumably mimicking the second P i product to dissociate, agrees with a position of the sulfate ion bound to the E. coli PPiase (PDB ID: 1JFD ).…”

Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei

“…Soluble PPases comprise two families that differ in both sequence and structure (10,11). Family/class I PPases occur in all types of cells from bacteria to humans, whereas class II occurs exclusively in bacteria (11)(12).…”