Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase-pyrophosphatases that act via a covalent aspartyl-phosphate intermediate.

Publication Type:

Journal Article


J Biol Chem, p.101851 (2022)


<p>Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phospho-substrate preferences. Here we interrogated the activities and structures of two DUF89 paralogs from fission yeast - Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and inorganic pyrophosphate. Crystal structures of metal-free Duf89 and Co-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl and NaF in the presence of divalent cations Co, Ni, or Zn generated covalent Duf8901-(Asp248)-BeF&bull;Co, Duf8901-(Asp248)-BeF&bull;Ni, or Duf8901-(Asp248)-BeF&bull;Zn adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)-phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF&bull;Co squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)-AlF-OH&bull;Co transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was de-repressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.</p>

Duf89, Duf8901•Co2+, Duf8901–BeF3•Co2+, Duf8901–BeF3•Ni2+, Duf8901–BeF3•Zn2+, and Duf8901–AlF4•Co2+ have been deposited in the RCSB protein structure databank under PDB ID codes 7T7N, 7T7K, 7T7O, 7U1V, 7U1X, and 7U1Y