Protonation states of the tryptophan synthase internal aldimine active site from solid-state NMR spectroscopy: direct observation of the protonated Schiff base linkage to pyridoxal-5'-phosphate

Abstract

The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional (13)C, (15)N, and (31)P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.

Figure 1

Schematic of PLP covalently bound to βLys87 in the β-subunit active site of tryptophan synthase. Distances and water molecules are from the X-ray crystal structure of the S. typhimurium TS internal aldimine state (PDB code 4HT3). Possible sites of protonation/ionization on the coenzyme and linking imine nitrogen are indicated in gray, along with hydrogen bonds to active-site residues/water. The donor and acceptor atoms of these hydrogen bonds are able to switch roles (potentially requiring another proton) to stabilize multiple, alternate protonation states, including the deprotonated Schiff base nitrogen/protonated phenolic oxygen pairing and/or protonation at the pyridine nitrogen (N1). The experimentally determined protonation states from solid-state NMR spectroscopy are highlighted in red. Protein residue fragments are shown in blue and PLP and water in black.

Figure 2

¹⁵N SSNMR cross-polarization magic-angle-spinning (CPMAS) spectra of the tryptophan synthase internal aldimine complex used to assign ¹⁵N chemical shifts to the linking Schiff base nitrogen (202.3 ppm) and N1 of PLP (294.7 ppm). Data were acquired on microcrystalline samples of S. typhimurium TS prepared with (A) TS at natural-abundance isotopomer concentration, (B) ε-¹⁵N-Lys TS, (C) natural-abundance TS/2,2′,3-¹³C₃;¹⁵N PLP, and (D, E) U-¹⁵N TS/2,2′,3-¹³C₃;¹⁵N PLP. (A–C) correspond to direct observation after cross-polarization from ¹H to ¹⁵N, while (D) and (E) form an ¹⁵N{¹³C}-REDOR pair; both have a 10 ms echo period after cross-polarization, but they differ in the application of π pulses to ¹³C (at the quarter and three-quarter mark of each rotor period) in (E). Experiments were performed at 9.4 T (400.37 MHz for ¹H, 100.69 MHz for ¹³C, 40.57 MHz for ¹⁵N) on a Bruker AVIII spectrometer equipped with an ¹H–¹³C–¹⁵N triple-resonance 4 mm MAS probe spinning at a MAS rate of 8 kHz and with the bearing gas cooled to −15 °C, giving an effective sample temperature of −5 °C. Cross-polarization was accomplished at an ¹H spin-lock field of 45 kHz, an ¹⁵N spin-lock field of 37 kHz (ramped ±2 kHz), and a contact time of 2 ms; 85 kHz Spinal64 ¹H decoupling was used throughout. During the REDOR echo periods, 14 μs π pulses were applied to both ¹³C and ¹⁵N. Each spectrum consists of the sum of 81,920 transients acquired with a relaxation delay of 4 s, for a total acquisition time of 3 days 19 h; the REDOR S and S_o spectra were acquired in an interleaved fashion. ¹⁵N chemical shifts were referenced indirectly to NH₃(l) via an external solid-state sample of ¹⁵NH₄Cl calibrated under MAS conditions as described in the SI. For comparison, we found δ[NH₃(l)] = δ[¹⁵NH₄Cl(s)] + 39.3 ppm.