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abstracts
Inorganic phosphate is an essential component of many biological molecules and processes including cellular signaling, generation of metabolic energy, DNA and RNA, and membrane phospholipids. Organophosphonates, which contain a highly stable carbon-phosphorus bond, are widely used as herbicidal, chelation, anti-scale, and medicinal agents. PhnY and PhnZ consist of a new oxidative catabolic pathway that is employed by marine bacteria to use 2-aminoethylphosphonic acid as a source of inorganic phosphate. PhnZ is notable for catalyzing the oxidative cleavage of a carbon-phosphorus bond using Fe(II) and dioxygen (see figure), despite belonging to a large family of hydrolytic enzymes, the HD-phosphohydrolase superfamily. We have determined structures of PhnZ in complex with its substrate, (R)-2-amino-1-hydroxyethylphosphonate. The structure reveals PhnZ to have an active site containing two Fe ions bound by 4 histidines and 2 aspartates (see figure) that is strikingly similar to the carbon-carbon bond cleaving enzyme, myo-inositol-oxygenase. Site-directed mutagenesis and kinetic analysis with substrate analogues revealed the roles of key active site residues. A 5th histidine that is conserved in the PhnZ subfamily specifically interacts with the substrate 1-hydroxyl. The structure also revealed that PhnZ possesses a unique induced-fit mechanism whereby an active-site aspartate specifically recognizes the 2-amino group of the substrate and toggles the release of an aromatic residue from the active site, thereby creating space for molecular oxygen bind to the second Fe ion. Structural comparisons of PhnZ reveal an evolutionary connection between Fe(II)-dependent hydrolysis of phosphate esters and oxidative carbon-phosphorus or carbon-carbon bond cleavage, thus uniting the diverse chemistries that are found in the HD superfamily.
abstracts
The 2-oxoglutarate/Fe(II)-dependent oxygenases (2OG oxygenases) are a large family of proteins that share a similar overall three-dimensional structure and catalyze a diverse array of oxidation reactions. The Jumonji C (JmjC)-domain containing proteins represent an important subclass of the 2OG oxygenase family that typically catalyze protein hydroxylation; however, recently other reactions have been identified, such as tRNA modification. The E. coli gene, ycfD, was predicted to be a JmjC-domain containing protein of unknown function based on primary sequence. Recently YcfD was determined to act as a ribosomal oxygenase, hydroxylating an arginine residue on the 50S ribosomal protein L-16 (RL-16). We have determined the crystal structure of YcfD at 2.7 Å resolution, revealing that YcfD is structurally similar to known JmjC proteins and possesses the characteristic double stranded β-helix fold or cupin domain. Separate from the cupin domain, an additional globular module termed -helical arm mediates dimerization of YcfD. We further have shown that 2-oxoglutarate binds to YcfD using isothermal titration calorimetry and identified R140 and S116 as key 2OG binding residues using mutagenesis which, together with the iron location and structural similarity with other cupin family members, allowed identification of the active site. Structural homology to ribosomal assembly proteins combined with GST-YcfD pull-down of a ribosomal protein and docking of RL-16 to the YcfD active site support the role of YcfD in regulation of bacterial ribosome assembly. Furthermore, overexpression of YcfD is shown to inhibit cell growth signifying a toxic effect on ribosome assembly.
