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Nucleotide Pyrophosphatase/Phosphodiesterase (NPP)
The overall dimeric structure of NPP in Xanthomonas axonopodis pv. citri str. 306. This enzyme relies on the catalytic ability of 2 Zn2+ atoms in the catalytic core, which are shown in white.
Identifiers
EC no.	3.6.1.9
Databases
IntEnz	IntEnz view
BRENDA	BRENDA entry
ExPASy	NiceZyme view
KEGG	KEGG entry
MetaCyc	metabolic pathway
PRIAM	profile
PDB structures	RCSB PDB PDBe PDBsum
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Nucleotide Pyrophosphatase/Phosphodiesterase (NPP) is a class of dimeric enzymes that catalyze the hydrolysis of phosphate diester bonds. NPP belongs to the Alkaline Phosphatase (AP) superfamily of enzymes. The NPP family of enzymes includes seven isoforms, some of which prefer nucleotide substrates, some of which prefer phospholipid substrates, and others of which prefer substrates that have not yet been determined. In eukaryotes, NPP enzymes are located in the cell membrane and hydrolyze extracellular phosphate diesters to affect a wide variety of biological processes.

Structure

The catalytic site of NPP consists of a two-metal-ion (bimetallo) Zn²⁺ catalytic core. These Zn²⁺ catalytic components are thought to stabilize the transition state of the NPP phosphoryl transfer reaction.

A closer view of the active site of NPP, which is located on the surface of the subunit. The Zn²⁺ atoms of the bimetallo catalytic site are shown by white spheres.

Mechanism

NPP catalyses the nucleophilic substitution of one ester bond on a phosphodiester substrate. The mechanism by which this phosphoryl transfer reaction is catalyzed is an area of ongoing research, though several extreme possibilities exist. The first mechanistic possibility is a dissociative (elimination-addition) mechanism, in which the phosphoryl transfer would involve a metaphosphate intermediate. The transition state for this transfer is referred to as “loose” in the scientific literature. The second mechanistic possibility is a concerted mechanism, which is an S_N2 type of mechanism in which one bond is broken while another is simultaneous formed. The transition state of this S_N2 type mechanism is referred to as “synchronous.” The final mechanistic possibility for phosphoryl transfer is an associative (addition-elimination) mechanism, in which a pentavalent phosphorane intermediate is formed. Transition states for this type of mechanism are referred to as “tight.” These three cases represent archetypes for the reaction mechanism, and the actual mechanism could fall somewhere in between.

Promiscuity

Although NPP primarily catalyzes phosphodiester hydrolysis, the enzyme will also catalyze the hydrolysis of phosphate monoesters, though to a much smaller extent. NPP preferentially hydrolyzes phosphate diesters over monoesters by factors of 10²-10⁶, depending on the identity of the diester substrate. This ability to catalyze a reaction with a secondary substrate is known as enzyme promiscuity.
This characteristic means that NPP is able to share substrates with alkaline phosphatase (AP), another member of the Alkaline Phosphate superfamily. Alkaline phosphatase primarily hydrolyzes phosphate monoester bonds, but interestingly it shows some promiscuity towards hydrolyzing phosphate diester bonds, making it a sort of opposite to NPP. The active sites of these two enzymes show marked similarities, namely in the presence of nearly superimposable Zn²⁺ bimetallo catalytic centers. In addition to the bimetallo core, AP also has an Mg²⁺ ion in its active site.

Evolution

NPP belongs to the Alkaline Phosphatase superfamily, which is a group of evolutionarily related enzymes that catalyze phosphoryl and sulfuryl transfer reactions. This group includes phosphomonoesterases, phosphodiesterases, phosphoglycerate mutases, phophophenomutases, and sulfatases.