Human indole(ethyl)amine-N-methyltransferase (hINMT) catalyzed methylation of tryptamine, dimethylsulfide and dimethylselenide is enhanced under reducing conditions - A comparison between 254C and 254F, two common hINMT variants

Abstract

Phenylalanine and cysteine comprise common miss-sense variants (i.e., single nucleotide polymorphisms [SNPs]) at amino acid position 254 of the human indole(ethyl)amine-N-methyltransferase (hINMT). The phenylalanine variant, which occurs in linkage disequilibrium with two 3' UTR SNPs, has been reported to associate with elevated urine levels of trimethylselenonium (TMSe), the Se-methylated product of volatile dimethylselenide. hINMT allozymes expressing either cysteine (254C) or phenylalanine (254F) at position 254 were compared for enzyme activity (i.e., Km and Vmax) towards the INMT substrates tryptamine, dimethylsulfide (DMS) and dimethylselenide (DMSe) in vitro. The SNP 254C had a higher Vmax for DMS and tryptamine in the presence of reducing agent than in its absence. Conversely, Vmax for 254F was insensitive to the presence or absence of reducing agent for these substrates. SNP 254F showed a lower Km for tryptamine in the absence of reducing agent than 254C. No statistically significant difference in Vmax or Km was observed between 254C and 254F allozymes in the presence of reducing agent for DMSe, The Km values for DMSe methylation were about 10-fold (254C) or 6-fold (254F) more favorable than for tryptamine methylation with reducing agent present. These findings indicated that: 1) That phenylalanine at position 254 renders hINMT methylation of substrates DMS and tryptamine insensitive to a non reducing environment. 2) That human INMT harbors significant thioether-S-methyltransferase (TEMT) activity with a higher affinity for DMSe than tryptamine, 3) The reduction of a 44C/254C disulfide bond in hINMT that increases Vmax is proposed.

Fig 1. hINMT purified protein.

Coomassie stained SDS-PAGE of 254C and 254F allozymes used in the kinetic study indicating the purity and the apparent molecular size.

Fig 2. hINMT enzyme activity: 254C vs. 254F.

Comparison of hINMT-catalyzed methylated product formation for 254C (A,C,E) and 254F (B, D, F) (-/+) 15 mM DTT for the substrates tryptamine (A, B), and DMS (C,D). The results for DMSe (E,F) are shown with (+) DTT only. Indicated concentrations of substrates were co-incubated with 5 μg of 254C hINMT or 254F hINMT and 30 μM ^14-C S-adenosyl-methionine as described in Materials and Methods. Statistical analysis of results is described in Table 1.

Fig 3. Cleavage of the proposed disulfide bond, 44C/254C, by DTT in hINMT.

Left side: hINMT crystal structure prepared without reducing agent with the position of common miss-sense SNPs (D28N, M206V, G219E and C254 [note: C44 is not at a position of a common SNP]) red-highlighted. Right side: Reducing agent-induced cleavage of proposed disulfide bond causes an increase in 254C enzyme activity; N-terminal tandem alpha-helical regions blue-highlighted; N- and C-termini, green and blue highlighted respectively.