The non-canonical hydroxylase structure of YfcM reveals a metal ion-coordination motif required for EF-P hydroxylation

EF-P is a bacterial tRNA-mimic protein, which accelerates the ribosome-catalyzed polymerization of poly-prolines. In Escherichia coli, EF-P is post-translationally modified on a conserved lysine residue. The post-translational modification is performed in a two-step reaction involving the addition of a β-lysine moiety and the subsequent hydroxylation, catalyzed by PoxA and YfcM, respectively. The β-lysine moiety was previously shown to enhance the rate of poly-proline synthesis, but the role of the hydroxylation is poorly understood. We solved the crystal structure of YfcM and performed functional analyses to determine the hydroxylation mechanism. In addition, YfcM appears to be structurally distinct from any other hydroxylase structures reported so far. The structure of YfcM is similar to that of the ribonuclease YbeY, even though they do not share sequence homology. Furthermore, YfcM has a metal ion-coordinating motif, similar to YbeY. The metal ion-coordinating motif of YfcM resembles a 2-His-1-carboxylate motif, which coordinates an Fe(II) ion and forms the catalytic site of non-heme iron enzymes. Our findings showed that the metal ion-coordinating motif of YfcM plays an essential role in the hydroxylation of the β-lysylated lysine residue of EF-P. Taken together, our results suggested the potential catalytic mechanism of hydroxylation by YfcM.

Waltham, MA) and washed with 5% B for 3 minutes. Peptides were eluted at a flow rate of 2 l/min with an increasing linear gradient of B from 5% to 30% over 47 minutes. The column was subsequently washed with 90% B for 5 minutes and the system was equilibrated for 10 minutes prior to the following injection.
An LTQ Orbitrap XL mass spectrometer was used to identify both the modified and unmodified forms of the Escherichia coli EF-P protein. Peptides were ionized using a captive spray ionization source (Michrome Bioresources Inc., Auburn, CA) with an ionization voltage and capillary temperature of 2.0 kV and 175C, respectively. Positive ion data acquisition was performed in a data-dependent fashion, with the dynamic exclusion and preview modes enabled. The top-5 precursor ions were selected for fragmentation with dynamic exclusion settings as follows: repeat count = 2, repeat duration = 20 s, exclusion list size = 100, exclusion duration = 60 s and exclusion mass width of ±1.50 m/z. Precursor ions underwent CID fragmentation in the LTQ linear ion trap with a normalized collision energy (NCE) of 35%. The raw data were converted to mzXML files using MSConvert and searched with MassMatrix (1,2) against a UniProt E. coli K12 proteome concatenated with modified forms of the EF-P sequence. This search confidently identified the (R)--lysyl-lysine and (R)--lysyl-hydroxylysine forms of EF-P. Once the protein identities were obtained, the experiment was repeated using the precursor ion inclusion mass list containing the precursor masses of the target peptides in multiple charged states. The instrument was operated in the orbitrap-orbitrap mode, in which both the precursor and product ions were detected in the orbitrap MS. This method was used to validate the identity of the obtained high mass resolution and the accuracy of the precursor and fragment ions for the (R)--lysyl-lysine and (R)--lysyl-hydroxylysine EF-P. The precursor and fragment ion resolutions were set at 15,000 and 7,500, respectively. Peptides bearing either (R)--lysyl-lysine or (R)--lysyl-hydroxylysine at position 34 and either a normal or oxidized Met16 were identified and manually validated (Table S6).
The combination of chromatographic separation and unique fragment ions allowed for highly specific and selective identification of the four modified forms of EF-P.
Once highly confident precursorproduct ion transitions were determined, follow-up parallel reaction monitoring (PRM) experiments were performed on an LTQ linear ion trap (Thermo Scientific, Waltham, MA) (3). This experiment leverages the faster scan rate of the ion trap and allows for the estimation of the conversion of (R)--lysyl-lysine to (R)--lysyl-hydroxylysine EF-P.
Again, the peptides were ionized using a captive spray ionization source (Michrome Bioresources Inc., Auburn, CA) with an ionization voltage and capillary temperature of 2.0 kV and 200C, respectively. To perform the PRM experiments, a target mass inclusion list containing the multiple charge states of the precursor ions was used, as described above. The precursor ions were selected with an isolation width of 1.5 Da and fragmented via CID (NCE 35%), and all fragment ion masses were collected. To differentiate between lysine hydroxylation and methionine oxidation, extracted ion chromatograms (XIC) were produced from the +3 species containing unique product ion transitions ( Figure S3 and Table S6). Finally, the yield of (R)--lysyl-hydroxylysine EF-P was estimated by the relative comparison of the corresponding XIC peaks. Quantification was performed using Thermo Xcalibur version 2.0, with Genesis algorithm peak detection and smoothing of 5. Figure        The numbers in parentheses are for the last shell.