Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36

Abstract

Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf-Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me- H 2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-L-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an S N 2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.enzyme mechanism | transition state structure | histone methylation | kinetic isotope effects

Kinetic Isotope Effects and Transition State Structure for Human Phenylethanolamine N-Methyltransferase

“…This primary methyl - 14 C KIE is consistent with values reported for NSD2, 14 DNMT1, 15 and glycine N -methyltransferase (GNMT). 21 The large magnitude of this KIE indicates that methyl transfer for hPNMT proceeds via a rate-limiting S N 2 mechanism.…”

“…22 These isotope effects are in agreement with previous studies that support methyl transfer as the rate-limiting step of the PNMT reaction. 9,10 DFT calculations for NSD2 14 and DNMT1 15 suggest that the primary 36 S KIE on methyltransferase reactions is proportional to the loss of bond order between the carbon of the transferring methyl (C Me ) and the sulfur of SAM at the TS. The 36 S KIE for hPNMT (1.014 ± 0.005) is smaller than those measured for NSD2 (1.018 ± 0.008) 14 and DNMT1 (1.019 ± 0.006), 15 suggesting that hPNMT TS structure may retain somewhat greater C Me –S bond order.…”

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

“…Our experimental KIEs and BIEs revealed that the SET8-catalyzed methylation goes through an early, asymmetrical S N 2 TS with the long N-C distance of 2.35-2.40 Å and the short S-C distance of 2.00-2.05 Å. Poulin et al (59) recently reported KIEs of NSD2 and solved its TS structure. In comparison with the early S N 2 TS of SET8, the TS structure of NSD2 shows the later, asymmetrical S N 2 characters with a short N-C distance of 2.02-2.10 Å and a long S-C distance of 2.5 Å.…”

The Role of Nuclear Receptor–Binding SET Domain Family Histone Lysine Methyltransferases in Cancer

“…Similar to NSD1, the NSD2 post-SET domain is attached to the catalytic SET domain via an autoinhibitory loop region and inhibition is relieved upon nucleosome binding (Qiao et al 2011; Poulin et al 2016a). Furthermore, NSD2 has been reported to preferentially catalyze H3K36 dimethylation compared to H3K36 monomethylation (Li et al 2009b; Kuo et al 2011; Poulin et al 2016b). …”