Structural and Functional Characterization of a Novel Phosphodiesterase from Methanococcus jannaschii

Abstract

Methanococcus jannaschii MJ0936 is a hypothetical protein of unknown function with over 50 homologs found in many bacteria and Archaea. To help define the molecular (biochemical and biophysical) function of MJ0936, we determined its crystal structure at 2.4-Å resolution and performed a series of biochemical screens for catalytic activity. The overall fold of this single domain protein consists of a four-layered structure formed by two ␤-sheets flanked by ␣-helices on both sides. The crystal structure suggested its biochemical function to be a nuclease, phosphatase, or nucleotidase, with a requirement for some metal ions. Crystallization in the presence of Ni 2؉ or Mn 2؉ produced a protein containing a binuclear metal center in the putative active site formed by a cluster of conserved residues. Analysis of MJ0936 against a panel of general enzymatic assays revealed catalytic activity toward bis-p-nitrophenyl phosphate, an indicator substrate for phosphodiesterases and nucleases. Significant activity was also found with two other phosphodiesterase substrates, thymidine 5 -monophosphate p-nitrophenyl ester and p-nitrophenylphosphorylcholine, but no activity was found for cAMP or cGMP. Phosphodiesterase activity of MJ0936 had an absolute requirement for divalent metal ions with Ni 2؉ and Mn 2؉ being most effective. Thus, our structural and enzymatic studies have identified the biochemical function of MJ0936 as that of a novel phosphodiesterase.Several hundred genomes have been sequenced so far, but no function can be inferred from the gene sequence of about half of the genes. Structural information may provide new clues as to the molecular (biochemical and biophysical) functions of the proteins coded by these genes of unknown functions.An open reading frame from Methanococcus jannaschii, MJ0936 (GI 1499771), is annotated as a conserved hypothetical protein in the PEDANT sequence data base (1). A PSI-PHI BLAST search found 56 bacterial or archaeal homologs with E value less than 0.003. Some of the homologs are annotated, and they can be grouped into three categories: (i) (conserved) hypothetical protein; (ii) VPS29-like (human vacuolar protein-sorting protein) phosphoesterase-related protein; (iii) predicted/ putative/probable phosphoesterase. Similarly, a Pfam (2) search suggests that this gene may belong to a protein family of calcineurin-like phosphoesterase. This Pfam protein family contains a large range of phosphoesterases, including protein phosphoserine phosphatases, nucleotidases, sphingomyelin phosphodiesterases, 2Ј,3Ј-cyclic AMP phosphodiesterases, and nucleases such as bacterial SbcD or yeast MRE11, as well as the VPS29-like phosphoesterase-related proteins mentioned above. Presumed metal-chelating residues are found to be conserved for the proteins of this family.To determine the function of MJ0936 and its homologues, we have solved the crystal structure of this protein and performed a series of biochemical screenings for catalytic activity. Through a combination of structural and enzymatic analyses, we ...

A Phosphate-binding Histidine of Binuclear Metallophosphodiesterase Enzymes Is a Determinant of 2′,3′-Cyclic Nucleotide Phosphodiesterase Activity

“…DR1281 resembles Rv0805 in its ϳ35-fold higher k cat for Mn 2ϩ -dependent hydrolysis of bis-p-nitrophenyl phosphate versus p-nitrophenyl phosphate and its selective hydrolysis of 2Ј,3Ј-cNMPs versus 3Ј,5Ј-cNMPs (23). The correlation between a nonhistidine residue and the absence of cyclic phosphodiesterase activity seen here with YfcE is reminiscent of the properties of the structurally characterized Methanococcus jannaschii enzyme MJ0936 (24). MJ0936 has vigorous activity in hydrolyzing bis-p-nitrophenyl phosphate but is unable to cleave p-nitrophenyl phosphate.…”

“…MJ0936 has vigorous activity in hydrolyzing bis-p-nitrophenyl phosphate but is unable to cleave p-nitrophenyl phosphate. Although possessed of a generic phosphodiesterase activity, MJ0936 reportedly had no detectable cyclic phosphodiesterase activity with the 2Ј,3Ј-or 3Ј,5Ј-forms of cAMP or cGMP (24). The crystal structure of manganese-bound MJ0936 (Protein Data Bank code 1S3N) (24) reveals the similarity of its active site to that of Rv0805, except for the presence of an asparagine in lieu of the phosphate-coordinating histidine.…”

Structure-based inference of molecular functions of proteins of unknown function from Berkeley Structural Genomics Center

“…The new structure is a "remote homologue," a structural homologue of a characterized protein structure despite the remoteness of its sequence similarity. BCSG examples of this category are MJ0882 from Methanococcus jannaschii (GI number 1499712) [4], MJ0936 from M. jannaschii (GI 1499771) [5], MG027 (GI 3844637) from M. genitalium [6], SP_1288 (GI 15675166) from Streptococcus pyogenes [7], MPN555 from M. pneumoniae (GI 1673958) [8], ScpB in Chlorobium tepidum (GI 21646405) [9], AQ_1354 (GI 2983779) from Aquifex aeolicu [10], PhoU proteins (GI 4982311) from Thermotoga maritima [11], YodA protein from E. coli (GI 16129919), TA1145 from Themoplasma acidophilum (GI 16082162) [12], R1281 from Deinococcus radiodurans (GI 6459028), and TM0651 (GI 4981173) from T. maritima [13]. All are the homologues of M. pneumoniae and M. genitalium proteins.…”

“…Since its crystal structure revealed structural homology to nucleases, phosphatases, and nucleotidases, a series of biochemical screens for a catalytic activity was performed and a novel phosphodiesterase activity was detected with an absolute requirement for divalent metal ions, Ni 2+ and Mn 2+ [5].…”

Structural Genomics of Minimal Organisms and Protein Fold Space

“…Its crystal structure, determined at 2.4 Å resolution (Figure 2), revealed structural homology to nucleases, phosphatases, or nucleotidases [14] with a Dali [15] Z-score higher than 6. A series of biochemical screens for catalytic activity was performed to test the biochemical activities suggested by the remote homologues.…”