Molecular basis for catabolism of the abundant metabolite trans-4-hydroxy-L-proline by a microbial glycyl radical enzyme
- Department of Chemistry, Massachusetts Institute of Technology, United States
- Department of Chemistry and Chemical Biology, Harvard University, United States
- Department of Biology, Massachusetts Institute of Technology, United States
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
Figures
Figure 1 with 2 supplements
Hyp dehydration is catalyzed by the GRE HypD in a prominent gut microbial metabolic pathway.
(A) Anaerobic microbial metabolism of trans-4-hydroxy-l-proline (Hyp) is catalyzed by Hyp dehydratase (HypD), a glycyl radical enzyme (GRE). The product of this transformation, (S)-Δ1-pyrroline-5-car…
Figure 1—figure supplement 1
Transformations catalyzed by GRE eliminases.
The moieties undergoing oxidation to drive elimination are highlighted in red. The eliminated functional group and the bond undergoing cleavage are highlighted in blue.
Figure 1—figure supplement 2
Enzymatic transformations involving 5-membered heterocyclic substrates.
The scaffolds undergoing oxidation to drive elimination are highlighted in red. The eliminated functional group and the bond undergoing cleavage are highlighted in blue.
Figure 2 with 3 supplements
Structure of C. difficile HypD with Hyp bound.
(A) Dimeric structure of HypD (green) with the glycyl radical domain that houses the Gly loop in yellow and the Cys loop in purple. Gly765, Cys434, and Hyp are shown in spheres. (B) 2Fo-Fc maps …
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Figure 2—source data 1
Cartesian coordinates for zwitterionic Hyp in Cγ-exo pucker calculated from DFT.
Coordinates of zwitterionic Hyp structure used to fit into the HypD crystal structure.
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Figure 2—source data 2
Cartesian coordinates for zwitterionic Hyp in Cγ-endo pucker calculated from DFT.
Coordinates of zwitterionic Hyp structure used to fit into the HypD crystal structure.
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Figure 2—figure supplement 1
A 2.05 Å resolution structure of HypD with glycerol bound in the active site.
(A) 2Fo-Fc maps (contoured at 1.0σ, gray) indicate electron density for glycerol. (B) Distances between glycerol hydroxyl groups and nearby residues are indicated. Water molecules are not shown.
Figure 2—figure supplement 2
HypD conformers generated by DFT calculations.
Structures of zwitterionic Hyp obtained for Cγ-endo and Cγ-exo puckered states. Cartesian coordinates are provided in source data.
Figure 2—figure supplement 3
Comparison of electron density maps for Cγ-exo Hyp versus Cγ-endo Hyp modeled into HypD active site.
2Fo-Fc maps (contoured at 1.0σ, gray) indicate electron density for substrate in both (A) Cγ-exo Hyp and (B) Cγ-endo Hyp pucker states. Conformers were restrained to the calculated optimal …
Figure 3 with 4 supplements
Active site of HypD has unique features that enable Hyp dehydration.
Conserved Gly and Cys loops in addition to active site residues are displayed for (A) HypD, (B) GD, and (C) CutC. PDB-deposited structures for GD (1R9D) and CutC (5FAU) were used to generate this …
Figure 3—figure supplement 1
Dihedral angles of substrates bound in GRE eliminases.
Dihedral angle between the departing group and the C1 hydroxyl group is displayed for (A) glycerol (GD, PDB: 1R9D), (B) (S)−1,2-propanediol, (PD, PDB: 5I2G), and (C) choline (CutC, PDB: 5FAU). …
Figure 3—figure supplement 2
Active sites of propanediol dehydratase (PD) and isethionate sulfite-lyase (IslA) compared to HypD.
Conserved Gly and Cys loops in addition to active site residues are displayed for (A) HypD, (B) PD, and (C) IslA. PDB-deposited structures for PD (5I2G) and IslA (5YMR) were used to generate this …
Figure 3—figure supplement 3
Proposed mechanism for CutC elimination reaction.
Ketyl radical intermediate for CutC is highlighted. Similar ketyl radical species have been proposed for GD and PD mechanisms.
Figure 3—figure supplement 4
A multiple sequence alignment of putative HypDs with characterized GREs.
A multiple sequence alignment of characterized GREs and putative HypDs selected to cover a wide range of phylogenetic diversity. Residues conserved among GRE dehydratases are highlighted in green. …
Figure 4
Hydrogen bonding and proline-aromatic interactions with nearby residues and bound water molecules allow for HypD chemistry.
(A) Residues and ordered water molecule (red sphere) that are within hydrogen bonding distance to the hydroxyl and amine of Hyp shown with corresponding distances. (B) Residues within hydrogen …
Figure 5 with 3 supplements
Most HypD variants do not have detectable activity.
(A) An in vitro coupled enzyme endpoint assay was used to measure activity of HypD variants. P5C generated from HypD activity was reduced to Pro by P5CR in assay mixtures. Pro and Hyp were …
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Figure 5—source data 1
Quantification of Pro and Hyp after HypD coupled assay using LC-MS/MS.
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Figure 5—source data 2
Source data for kinetic analysis of HypD-Y450F and HypD-T645A enzyme variants.
Individual data points for glycyl radical-normalized kobs values of HypD-Y450F and HypD-T645A variants in the HypD coupled assay (scheme shown in Figure 5—figure supplement 2A). These values were used to plot Michaelis-Menten kinetic curves in Figure 5—figure supplement 2B (HypD-Y450F) and Figure 5—figure supplement 2C (HypD-T645A). Figure 6—source data 1. LC-MS/MS data for HypD D2O assay and HypD assay using 2,5,5-D3-Hyp as substrate. LC-MS/MS and calculated percentages of total ions calculated for commercial standard of Pro diluted in D2O and for HypD coupled assays run in D2O, described in Figure 6. The mass 116.1 corresponds to the precursor undeuterated Pro ion, and the fragment 70.1 corresponds to the mass of Pro ion after fragmentation of the carboxylate group. These data were used to calculate average deuterium incorporation in Pro commercial standard in D2O and Pro generated by HypD reaction run in D2O, both presented in Figure 6C.
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Figure 5—figure supplement 1
Activation of HypD variants detected by EPR spectroscopy.
Glycyl radical formation was quantified by EPR spectroscopy for all HypD variants and wild-type. Representative EPR spectra are shown here for each variant. Both simulated (top trace) and …
Figure 5—figure supplement 2
Kinetic analysis of HypD-Y450F and HypD-T645A.
(A) HypD activity was coupled to P5CR and absorbance at 340 nm was measured to calculate initial rates for NADH consumption. (B) Michaelis–Menten kinetic curve using glycyl radical-normalized values …
Figure 5—figure supplement 3
SDS-PAGE of purified proteins used in this study.
(A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the proteins used in biochemical assays. Samples were loaded as follows: lane 1 and 11 – Precision Plus Protein All Blue …
Figure 6 with 3 supplements
Coupled HypD and P5CR assay with trideuterated substrate 2,5,5-D3-Hyp results in the formation of product 2,4,5-D3-Pro.
(A) Overall reaction scheme for HypD P5CR coupled assay with 2,5,5-D3-Hyp. (B) P5C nonenzymatically hydrolyzes to an aldehyde product that equilibrates between keto and enol tautomers. (C) HypD …
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Figure 6—source data 1
LC-MS/MS data for HypD D2O assay and HypD assay using 2,5,5-D3-Hyp as substrate.
LC-MS/MS and calculated percentages of total ions calculated for commercial standard of Pro diluted in D2O and for HypD coupled assays run in D2O, described in Figure 6. The mass 116.1 corresponds to the precursor undeuterated Pro ion, and the fragment 70.1 corresponds to the mass of Pro ion after fragmentation of the carboxylate group. These data were used to calculate average deuterium incorporation in Pro commercial standard in D2O and Pro generated by HypD reaction run in D2O, both presented in Figure 6C.
- https://cdn.elifesciences.org/articles/51420/elife-51420-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
1H NMR of Pro and HypD coupled assay product 2,4,5-D3-Pro.
1H NMR spectra are shown for (A) a commercial Pro standard and (B) 2,4,5-D3-Pro, the product of the HypD coupled assay. Peaks for carbons are labeled and color-coded according to the inset Pro and …
Figure 6—figure supplement 2
13C NMR of Pro and HypD coupled assay product 2,4,5-D3-Pro.
13C NMR spectra are shown for (A) a commercial Pro standard and (B) 2,4,5-D3-Pro, the product of the HypD coupled assay (including a zoomed in panel in the inset to better view peaks for C2-C5). …
Figure 6—figure supplement 3
COrrelated SpectroscopY (COSY) NMR of HypD coupled assay product 2,4,5-Pro.
COSY NMR show coupling between vicinal protons on C3 and C4, and between C4 and C5. Peaks for protons are labeled and color-coded according to the inset 2,4,5-D3-Pro shown.
Figure 7
Proposed mechanism for HypD dehydration of deuterated substrate 2,5,5-D3-Hyp.
Figure 8
Change in Hyp puckering throughout the mechanism is proposed to play a critical role in radical transfer.
(A) Cγ-exo puckering of Hyp positions the pro-S hydrogen atom of Hyp C5 in closest proximity to Cys434 for hydrogen atom abstraction. (B) The product P5C is modeled into Hyp-bound HypD structure by …
Tables
Table 1
Data collection and model refinement statistics for crystallography.
Values in parentheses denote highest resolution bin.
| HypD with glycerol bound | HypD with Hyp bound | |
|---|---|---|
| Space group | P21 | P21 |
| Unit cell (Å) | 100.3, 341.7, 122.6, 90.0°, 107.1°, 90.0° | 101.2, 350.2, 124.5, 90.0°, 105.7°, 90.0° |
| Resolution (Å) | 50–2.05 (2.09–2.05) | 50–2.52 (2.59–2.52) |
| Rsym | 16.8 (75.7) | 20.4 (97.5) |
| CC1/2 | 99.0 (58.8) | 99.4 (72.1) |
| <I/σ> | 8.40 (1.82) | 10.75 (2.12) |
| Completeness (%) | 99.0 (98.3) | 99.7 (99.4) |
| Unique reflections | 486251 (24062) | 278476 (44812) |
| Total reflections | 1626596 (168674) | 1944676 (294711) |
| Redundancy | 7.07 (7.01) | 6.98 (6.58) |
| Rwork/Rfree | 0.166/0.193 | 0.186/0.224 |
| RMSD bond length (Å) | 0.007 | 0.008 |
| RMSD bond angles (°) | 0.86 | 0.966 |
| Chains in asymmetric unit | 8 | 8 |
| Number of: | ||
| Total atoms | 54954 | 52103 |
| Protein atoms | 49994 | 49851 |
| Water molecules | 4834 | 2180 |
| Gol/Hyp | 48 | 72 |
| Ramachandran analysis | ||
| Favored (%) | 98.16 | 97.71 |
| Allowed (%) | 1.71 | 1.99 |
| Disallowed (%) | 0.13 | 0.30 |
| Rotamer outliers (%) | 1.46 | 3.27 |
| Average B factors | ||
| Protein (Å2) | 21.0 | 35.8 |
| Water (Å2) | 27.2 | 27.7 |
| Gol/Hyp (Å2) | 22.3 | 31.2 |
Table 2
Glycyl radical quantification, activity, and kinetic parameters for HypD variants.
Mean and SD are displayed for glycyl radical quantification where n = 3 independent experiments for each protein. HypD activity was coupled to P5CR and absorbance at 340 nm was measured to calculate …
| HypD | Radical per monomer (%) | Activity detected by quantification of proline | Km (mM) | Un-normalized kcat (s−1) | Glycyl radical- normalized kcat (s−1) | Catalytic efficiency using normalized kcat (M−1 s−1) |
|---|---|---|---|---|---|---|
| Wildtype (Levin et al., 2017) | 51 ± 1 | Yes | 1.2 ± 0.1 | 46 ± 1 | 45 ± 1 | 3.8 ± 0.3 × 104 |
| G765A | 0 | No | ND | ND | ND | |
| C434S | 34 ± 8 | No | ND | ND | ND | |
| E436Q | 12.4 ± 0.5 | No | ND | ND | ND | |
| H160Q | 4.4 ± 0.8 | No | ND | ND | ND | |
| D278N | 16 ± 4 | No | ND | ND | ND | |
| D339N | 18 ± 8 | No | ND | ND | ND | |
| S334A | 50 ± 19 | No | ND | ND | ND | |
| Y450F | 29 ± 4 | Yes | 19 ± 3 | 0.33 ± 0.01 | 0.57 ± 0.04 | 30 ± 6 |
| T645A | 19 ± 1 | Yes | 4.9 ± 0.4 | 0.75 ± 0.01 | 1.98 ± 0.04 | 400 ± 30 |
| Y450F/T645A | 3.4 ± 0.8 | No | ND | ND | ND | |
| F340A | 23 ± 5 | No | ND | ND | ND |
Table 3
Primers used in site-directed mutagenesis of HypD.
Nucleotides mutated are indicated in small letters.
| Primer | Sequence (5′ to 3′) | Annealing temperature used, °C |
|---|---|---|
| pET28a-CdHypD-G765A-fwd | GACTTAATAGTTAGAGTTGCAGcATATAGTGACCATTTC | 66 |
| pET28a-CdHypD-G765A-rev | CTACTTAAATTATTGAAATGGTCACTATATgCTGCAACTCTAAC | 66 |
| pET28a-CdHypD-C434S-fwd | AACCAGTGGTTcTGTTGAAACTGGATG | 58 |
| pET28a-CdHypD-C434S-rev | CAGTTTCAACAgAACCACTGGTTCCACC | 58 |
| pET28a-CdHypD-E436Q-fwd | CAGTGGTTGTGTTcAAACTGGATGTTTTGG | 60 |
| pET28a-CdHypD-E436Q-rev | ACATCCAGTTTgAACACAACCACTGGTTC | 60 |
| pET28a-CdHypD-H160Q-fwd | AGCCCCAGGACAgACAGTTTGTGGAGATAC | 60 |
| pET28a-CdHypD-H160Q-rev | ACAAACTGTcTGTCCTGGGGCTCTTTGTTC | 60 |
| pET28a-CdHypD-D278N-fwd | GAACTTAATATATGGaATGCTTTTACTCCAGGAAGACTTGACC | 66 |
| pET28a-CdHypD- D278N-rev | CCTGGAGTAAAAGCATtCCATATATTAAGTTCAGTAGTAACCCC | 66 |
| pET28a-CdHypD- F340A-fwd | GAAAGTAGCACATATACAGATgcTGCAAATATAAAC | 54 |
| pET28a-CdHypD- F340A-rev | GATTTATTCCACCAGTGTTTATATTTGCAgcATCTGTATATG | 54 |
| pET28a-CdHypD- Y450F-fwd | GTTTTGGTAAAGAAGCATATGTTCTAACTGGATtTATGAACATTCC | 66 |
| pET28a-CdHypD- Y450F-rev | GTATTTTTGGAATGTTCATAaATCCAGTTAGAACATATGCTTCTTTACC | 66 |
| pET28a-CdHypD- S334A-fwd | GTTGGTATAACATTAAAAGAAgcTAGCACATATACAGATTTTGC | 60 |
| pET28a-CdHypD- S334A-rev | CTGTATATGTGCTAgcTTCTTTTAATGTTATACCAACTTTTGG | 60 |
| pET28a-CdHypD- T645A-fwd | ATGTTACCAgCAACTTGTCATATATACTTTGGAGAAATTATGGG | 66 |
| pET28a-CdHypD- T645A-rev | TATGACAAGTTGcTGGTAACATATCTACTCTGTATTCTCCACC | 66 |
| pET28a-CdHypD- D339N-fwd | CATTAAAAGAAAGTAGCACATATACAaATTTTGCAAATATAAACACTGG | 66 |
| pET28a-CdHypD- D339N-rev | GGATTTATTCCACCAGTGTTTATATTTGCAAAATtTGTATATGTGCTAC | 66 |
