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 (His21 and His86) in the active site. The 1.3-A three-dimensional structure of mtPPase shows that His86 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 Mg2+, but it differs from the E. coli enzyme and family II PPases because it has much lower activity in the presence of Mn2+ or Zn2+. Replacements of His21 and His86 in mtPPase with the residues found in the corresponding positions of E. coli PPase had either no effect on the Mg2+- and Mn2+-supported reactions (H86K) or reduced Mg2+-supported activity (H21K). However, both replacements markedly increased the Zn2+-supported activity of mtPPase (up to 11-fold). In the double mutant, Zn2+ was a 2.5-fold better cofactor than Mg2+. These results show that the His residues in mtPPase are not essential for catalysis, although they determine cofactor specificity.
Highlights
Family I PPases, which are fairly widespread in all types of organisms, and family II PPases, which are exclusive to bacteria
There are a total of three such histidine residues, two act as ligands that bind to metal ions and one is believed to interact with bound PPi [6, 7]
We present the structure of a family I PPase from Mycobacterium tuberculosis, which closely resembles the structure of other bacterial family I PPases but is unique in having two histidine residues (His21 and His86) in the active site
Summary
Cloning of mtPPase—The Rv3628 gene encoding mtPPase was kindly provided by Dr Florence Proux (Institut Pasteur, Paris) on a pDEST17 vector and was amplified by PCR using: 5Ј-CACCACCACCACATGCAATTCGACGTGACCATCGAAA-3Ј as forward primer and 5Ј-GAGGAGAAGGCGCGTTATCAGTGTGTACCGGCCTTGAAGCGCTC-3Ј as a reverse primer. Expression and Purification of mtPPase—The mtPPase-pET-YSBLIC plasmid was first transformed into NovaBlue singles (Novagen) for plasmid production and into E. coli BL21(DE3) cells for protein expression. Fractions containing mtPPase were pooled (50 ml), dialyzed overnight against 40 volumes of 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, concentrated to 5 ml by ultrafiltration using Vivaspin concentrators (Vivascience, Sartorius group), and loaded onto a Superdex 200 16/60 gel filtration column that had been washed with Tris-HCl buffer. Model building was completed using the program COOT [22], and further cycles of refinement were carried out with REFMAC coupled with Arp/wArp to update the water molecules. The model was refined again without an automatic water update to a final Rfactor of 15.4% and Rfree of 16.9%. The dependence of kcat on metal cofactor concentration were fit to Equation 4, where [M] is the cofactor concentration, KM is the dissociation constant of the enzyme-metal complex, and kcat,lim is the limiting value of kcat at saturating [M] as shown in the following equations
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