title: Leucine 208 in human histamine N-methyltransferase emerges as a hotspot for protein stability rationalizing the role of the L208P variant in intellectual disability.
abstract: The degradation of histamine catalyzed by the SAM-dependent histamine N-methyltransferase (HNMT) is critically important for the maintenance of neurological processes. Recently, two mutations in the encoding human gene were reported to give rise to dysfunctional protein variants (G60D and L208P) leading to intellectual disability. In the present study, we have expressed eight L208 variants with either apolar (L208F and L208V), polar (L208N and L208T) or charged (L208D, L208H, L208K and L208R) amino acids to define the impact of side chain variations on protein structure and function. We found that the variants L208N, L208T, L208D and L208H were severely compromised in their stability. The other four variants were obtained in lower amounts in the order wild-type HNMT>L208F=L208V>L208K=L208R. Biochemical characterization of the two variants L208F and L208V exhibited similar Michaelis-Menten parameters for SAM and histamine while the enzymatic activity was reduced to 21% and 48%, respectively. A substantial loss of enzymatic activity and binding affinity for histamine was seen for the L208K and L208R variants. Similarly the thermal stability for the latter variants was reduced by 8 and 13°C, respectively. These findings demonstrate that position 208 is extremely sensitive to side chain variations and even conservative replacements affect enzymatic function. Molecular dynamics simulations showed that amino acid replacements in position 208 perturb the helical character and disrupt interactions with the adjacent β-strand, which is involved in the binding and correct positioning of histamine. This finding rationalizes the gradual loss of enzymatic activity observed in the L208 variants.
authors: Graz University of Technology, Institute of Biochemistry, Petersgasse 12/II, A-8010 Graz, Austria.
authors: Chanakan
authors: C
authors: Tongsook
authors: Graz University of Technology, Institute of Biochemistry, Petersgasse 12/II, A-8010 Graz, Austria.
authors: Johannes
authors: J
authors: Niederhauser
authors: Graz University of Technology, Institute of Biochemistry, Petersgasse 12/II, A-8010 Graz, Austria.
authors: Elena
authors: E
authors: Kronegger
authors: Graz University of Technology, Institute of Biochemistry, Petersgasse 12/II, A-8010 Graz, Austria.
authors: Grit
authors: G
authors: Straganz
authors: Graz University of Technology, Institute of Biochemistry, Petersgasse 12/II, A-8010 Graz, Austria. Electronic address: peter.macheroux@tugraz.at.
authors: Peter
authors: P
authors: Macheroux
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006632:Histamine / Q000378:metabolism; D006637:Histamine N-Methyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics* / Q000378:metabolism; D007930:Leucine / Q000235:genetics* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D017354:Point Mutation; D011487:Protein Conformation; D055550:Protein Stability; D016415:Sequence Alignment
chemical_list: D006632:Histamine; D006637:Histamine N-Methyltransferase; D007930:Leucine
keywords: Isothermal titration calorimetry; Molecular dynamics simulations; Neurotransmitter; Protein stability; S-adenosylmethionine