Mutations in the Different Residues between Dextransucrase Gtf-DSM and Reuteransucrase GtfO for the Investigation of Linkage Specificity Determinants

Abstract

The dextransucrase Gtf-DSM has 99.3% sequence identity with the reuteransucrase GtfO, and only 11 out of 1045 residues are different between their N-terminally truncated recombinant forms. Gtf-DSM is capable of synthesizing a dextran with 1% (α1 → 2), 6% (α1 → 4), 24% (α1 → 3), and 69% (α1 → 6) linkages, while GtfO produces a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. In this work, using recombinant Gtf-DSM and GtfO as templates, parallel substitutions targeting these 11 distinguishing residues were performed to investigate their linkage specificity determinants. The combinatorial mutation (I937L/D977A/D1083V/Q1086K/K1087G) at the acceptor binding subsites +1 and +2 nearly converted the linkage specificity of Gtf-DSM to that of GtfO. Surprisingly, all of the individual or combinatorial mutations in four residues from domains IV and V of Gtf-DSM significantly altered the linkage specificity of Gtf-DSM. Additionally, all mutations in the 11 distinguishing residues of Gtf-DSM resulted in a dramatically reduced transferase/hydrolysis activity ratio, which was closer to that of GtfO. These mutation results suggested that the linkage specificity differences between Gtf-DSM and GtfO are determined by the distinct micro-physicochemical environments, formed by the concerted action of a series of residues not only from the acceptor binding subsites +1 and +2 but also from domains IV and V.

Insights into the Unusual Activity of a Novel Homospermidine Synthase with a Promising Application to Produce Spermidine

“…Utilized were the Charmm36 force field and the TIP3P water model. The energy-minimized structures were subjected to 100 ps canonical ensemble (NVT) and 100 ps isothermal–isobaric ensemble (NPT) simulations at 318 K and 1 bar pressure . All bonds were constrained by the LINCS algorithm, and the total simulation time was 100 ns; conformations were saved every 10 ps.…”

“…The energy-minimized structures were subjected to 100 ps canonical ensemble (NVT) and 100 ps isothermal−isobaric ensemble (NPT) simulations at 318 K and 1 bar pressure. 28 All bonds were constrained by the LINCS algorithm, and the total simulation time was 100 ns; conformations were saved every 10 ps. After the simulation was completed, the simulated trajectories were analyzed using VMD.…”