Structural Insight into the Mechanism of Substrate Specificity and Catalytic Activity of an HD-Domain Phosphohydrolase: The 5′-Deoxyribonucleotidase YfbR from Escherichia coli

Abstract

HD-domain phosphohydrolases have nucleotidase and phosphodiesterase activities and play important roles in the metabolism of nucleotides and in signaling. We present three 2.1-A-resolution crystal structures (one in the free state and two complexed with natural substrates) of an HD-domain phosphohydrolase, the Escherichia coli 5'-nucleotidase YfbR. The free-state structure of YfbR contains a large cavity accommodating the metal-coordinating HD motif (H33, H68, D69, and D137) and other conserved residues (R18, E72, and D77). Alanine scanning mutagenesis confirms that these residues are important for activity. Two structures of the catalytically inactive mutant E72A complexed with Co(2+) and either thymidine-5'-monophosphate or 2'-deoxyriboadenosine-5'-monophosphate disclose the novel binding mode of deoxyribonucleotides in the active site. Residue R18 stabilizes the phosphate on the Co(2+), and residue D77 forms a strong hydrogen bond critical for binding the ribose. The indole side chain of W19 is located close to the 2'-carbon atom of the deoxyribose moiety and is proposed to act as the selectivity switch for deoxyribonucleotide, which is supported by comparison to YfdR, another 5'-nucleotidase in E. coli. The nucleotide bases of both deoxyriboadenosine-5'-monophosphate and thymidine-5'-monophosphate make no specific hydrogen bonds with the protein, explaining the lack of nucleotide base selectivity. The YfbR E72A substrate complex structures also suggest a plausible single-step nucleophilic substitution mechanism. This is the first proposed molecular mechanism for an HD-domain phosphohydrolase based directly on substrate-bound crystal structures.

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

“…S13) comprises entirely uncharacterized proteins. This clade is more closely related to PhnZ than to the known hydrolases (HD-GYP and YfbR), its members are missing the aforementioned Arg residue of YfbR, and a biochemical screen for phosphohydrolase/nucleotidase activity found E. coli YedJ to lack such activity (9). Thus, these proteins are more likely MVDOs rather than phosphohydrolases, as they have been annotated.…”

“…S12), but they are very likely to be nucleotidases, because two different structures (PDB ID codes 2OGI and 2O08) have (d)NDPs bound to the dimetal centers. These proteins have a conserved Arg residue in a position spatially equivalent to that occupied by a functionally essential Arg in YfbR and other mononuclear HD phosphohydrolases such as the HIV-restricting triphosphohydrolase SAMHD1 (9,12). The corresponding residues in MIOX and PhnZ are Asn and Gln.…”

“….D region of the domain form a second metal binding site, have also been identified (6,7). Only a limited number of HD proteins have been biochemically characterized; they almost exclusively catalyze phosphoester hydrolysis, which has led to the general assignment of HD proteins as phosphohydrolases (3,5,(8)(9)(10)(11)(12). The wide phylogenetic distribution and diverse domain architectures of the HD proteins suggest, however, that additional activities might have evolved within the superfamily.…”

An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis

“…This superfamily is divided into classes of enzymes that use a His-Asp doublet of residues and an additional series of conserved His and Asp residues to coordinate a single divalent metal (HX n HDX n D motif) (8,9), a dinuclear metal active site (HX n HDX n HX n HX n D motif) (10,11), or a recently described trinuclear iron metal-center (12). Although in many cases the identity of the relevant catalytic metal required for chemistry in vivo is unclear, there are examples of mononuclear enzymes that can use magnesium (13), cobalt (14), or manganese (8,13,15), and dinuclear enzymes that can use nickel (16), manganese (17), or iron (10,(18)(19)(20).…”

“…Their reactions are reasonably diverse and include hydrolysis of a phosphate from 2′-deoxyadenosine monophosphate (dAMP) by the Escherichia coli enzyme YfbR (14), hydrolysis of pyrophosphate from (p)ppGpp (13) or a deoxyribonucleoside triphosphate (15), and hydrolysis of inorganic triphosphate from deoxyadenosine triphosphate (dATP) (21,22). These enzymes can also cleave the phosphodiester bond of cyclicdi-GMP (11,23).…”