Enhancing Human Spermine Synthase Activity by Site Directed Mutations

Abstract

Spermine Synthase (SMS) is an enzyme converting spermidine into spermine, both of which are polyamines controlling normal cell growth and development. Several missense mutations in human SMS (HsSMS) are known to cause Snyder-Robinson Syndrome (SRS) by either destabilizing the monomer/dimer conformation or directly affecting the hydrogen bond network in the active sites. Recently a comparison of protein sequence and crystal structure between the HsSMS and its homologous protein Thermotoga maritima (Tm) spermidine synthase (TmSRM) was performed. Tm is the only bacterium known to grow at a high temperature as well as 90°C, and the half-life of TmSRM is longer than 25h under this temperature. In contrast, HsSMS is much less stable than TmSRM under the same temperature. Sequence alignment between HsSMS and TmSRM suggests that some key residues may be essential players for the elevate stability of TmSRM. Such key residues were identified based on various biophysical and sequence criteria and four mutations (S165D, L175E, T178H and C206R) were selected for HsSMS. Both in silico and in vitro experiments indicated that these four mutations strongly stabilize the monomer structure and dramatically improve the efficiency of SPM synthesis. The enhanced reaction rate in the mutant HsSMS is attributed to the increase of the strength of negative electrostatic potential, calculated with DelPhi, in the dimer cleft between HsSMS units, which presumably facilitates the substrate delivery to the active site.The work was supported by a grant from the Institute of General Medical Sciences, National Institutes of Health, and the grant number is 1R01GM093937.