Supplementary Data

Biosynthesis of the 22nd Genetically Encoded Amino Acid,

Pyrrolysine: Structure and Reaction Mechanism of PylC at 1.5 Å

Resolution

Felix Quitterer, Anja List, Philipp Beck, Adelbert Bacher, and Michael Groll

Fig. S1. Structural superposition of the D1 domain of PylC from M. barkeri (blue) and the

corresponding N-terminal domain of BC from Pseudomonas aeruginosa (orange) as stereo

presentation. The red arrow indicates that the ATP molecule can only bind in the PylC structure.

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Fig. S2. Structural superposition of the D2 domain of PylC from M. barkeri (blue) and the

corresponding central domains of Ddl from Thermus thermophilus (green) and BC from

Campylobacter jejuni (orange) as stereo presentation. The T-loop of PylC shown in red is not

structurally constrained (in contrast to Ddl and BC, indicated by a red arrow) and thus is able to

perform simple structural rearrangements without causing major domain movements.

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Fig. S3. Amino acid residues in contact with both adenylates (black) and LysN-D-Orn (ornithyl

moiety: green, lysyl moiety: yellow) from the crystal structure of state IV. The interaction

distances are shown in Å. In the upper right corner the octahedral coordination of both Mg 2+ ions

is displayed.

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Fig. S4. Stereo view of the active site of PylC representing state IV*, harboring the modeled

natural PylC substrate, 3MO. The 2Fo-Fc omit electron density map (blue mesh) is shown from

the crystal structure displaying state IV and is contoured at 1.0 σ. Color coding and orientation

are according to Fig. 4. Note, the electron density map displays an alternative conformation of the

D-Orn side chain in which the methyl group perfectly fits and performs Van-der-Waals

interactions with V187 (indicated by a red arrow).

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Fig. S5. Primary sequence alignment of PylC proteins. Secondary structure elements are shown

for PylC. Strictly conserved amino acid residues in contact with LysN-D-Orn are highlighted in

blue, functionally conserved residues are highlighted in light blue. Strictly or functionally

conserved amino acid residues not in contact with the product are shown in grey and light grey,

respectively. METBF: Methanosarcina barkeri Fusaro, METBA: Methanosarcina barkeri,

METBU: Methanococcoides burtonii, 9FIRM: Desulfosporosinus orientis, DESHA:

Desulfitobacterium hafniense, METMA: Methanosarcina mazei, ACEAZ: Acetohalobium

arabaticum, DESAS: Desulfotomaculum acetoxidans, METAC: Methanosarcina acetivorans,

METEZ: Methanohalobium evestigatum, THEPJ: Thermincola potens, METZD: Methanosalsum

zhilinae, METMS: Methanohalophilus mahii, BILWA: Bilophila wadsworthia.

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Fig. S6. Primary sequence alignment of PylC from Methanosarcina barkeri Fusaro with Ddl

from Thermus thermophilus HB8. Secondary structure elements are shown for PylC and Ddl,

residue numbers are displayed for PylC. Color coding of the letter background refers to PylC and

is according to Fig. S5. Strictly conserved residues for PylC and Ddl are shown in bold red

letters.

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Fig. S7. Stereo view of Fig. 3a.

Fig. S8. Stereo view of Figs. 8a and 8b.

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Supplementary References

1. Mullins, L. S., Zawadzke, L. E., Walsh, C. T. & Raushel, F. M. (1990). Kinetic evidence

for the formation of D-alanyl phosphate in the mechanism of D-alanyl-D-alanine ligase. J.

Biol. Chem. 265, 8993-8998.

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