Identification of the High Affinity Mn²⁺ Binding Site of Bacteriophage λ Phosphoprotein Phosphatase:  Effects of Metal Ligand Mutations on Electron Paramagnetic Resonance Spectra and Phosphatase Activities

Abstract

Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to lambdaPP are E. coli PrpA and PrpB. Using the crystal structure of lambdaPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics. Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and [(Mn(2+))(2)] binding. The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…The second metal ion may enter the active site upon substrate binding, thus, reconstituting a fully active enzyme. 135 A similar mechanism has also been proposed for the proofreading activity of DNA polymerases (see section 4).…”

“…112 site has a homobinuclear Mn(II)-Mn(II) center. 134,135 Similar to PAPs, the two metal binding sites are distinguished by considerable differences in their affinities for the metal ions, with K D values of ∼2 and ∼160 µM, respectively. 134 A recent redox titration study of the Fe-Zn derivative of λPP has determined the redox potential for the Fe(III)/Fe(II) couple to be ∼120 mV.…”

“…Similar to PAPs, the two metal centers can be distinguished with respect to their metal ion affinities. 135 However, in contrast to PAPs, λPP displays a higher affinity in site M2 (K D (MnII) ∼ 2 µM).…”

Mechanism of the phosphatase component of Clostridium thermocellum polynucleotide kinase-phosphatase

“…Mutational studies of l phosphatase had established essential catalytic roles for five metal-binding residues: Asp20, His22, Asp49, Asn75, and His186 (Zhuo et al 1994;White et al 2001), which are equivalent to Asp187, His189, Asp233, Asn263, and His376 of CthPnkp. Of particular interest is the essential Asn263 side chain of CthPnkp, which is consistently seen to make contacts to both manganese (via Od) and one of the phosphate oxygens (via Nd) (Figs.…”

The UDP-diacylglucosamine Pyrophosphohydrolase LpxH in Lipid A Biosynthesis Utilizes Mn2+ Cluster for Catalysis

“…These enzymes perform catalysis using an ordered shell of water molecules and divalent or trivalent metal cofactors coordinated by conserved residues of the metallophosphoesterase motif (11,12). It has been postulated that the metal ions serve to bridge an oxide that performs a nucleophilic attack, to stabilize the formation of a phosphorane intermedi-ate, and to coordinate a nucleophilic hydroxyl group to allow for deprotonation (12,13). Besides calcineurin, enzymes of the CLP superfamily also include protein serine/threonine phosphatases (14), purple acid phosphatases (15), nucleotidases, sphingomyelin phosphodiesterases (16), exonucleases, and cyclic nucleotide phosphodiesterases (11).…”