Part:BBa_K3647666

pT7-Histamine N-methyltransferase (HNMT)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 109
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 463
  Illegal BsaI.rc site found at 847
  

General information

Histamine N-methyltransferase (HNMT) is one of the two enzymes involved in the metabolism of histamine in mammals, this is known as the N(tau)-methylation pathway. The second pathway is oxidative deamination via diamine oxidase (DAO). This protein is encoded by the gene HNMT and it can be found in the cytosol intracellular fluid. HNMT is expressed in many organs like the liver, kidney, and brain, is found in most of the body tissues, except for serum. (Yoshikawa, 2019)

The function of HNMT is catalyzation of histamine methylation in the presence of S-adenosylmethionine (SAM-e) yielding N-methylhistamine. Histamine (HA) is inactivated by using S-adenosylmethionine (SAM-e) as a methyl donor. This occurs, as an example, during the termination process of neurotransmission actions of HA in the mammalian central nervous system. (Heidari et al, 2015) (Yoshikawa, 2019)

Figure 1. Structure of histamine N-methyltransferase. PBD code 1JQD.

Human Histamine N-methyltransferase is a monomeric protein. It consists of 292 amino acids, of chains A and B, and has a weight of 33,33 kDa. It is a two-domain structure, the large domain is an MTase fold, defined by 20 MTases and the S domain. The MTase domain is the one that participates in histamine binding. The polymorphic amino acid 105 is located on the outer surface of the MTase domain. The HNMT gene is located on chromosome 2q22.1 and has six exons of 50kb.(Horton, 2001)

HNMT is released via mast cells degranulation upon bronchoconstriction thus contributing to the asthmatic symptoms. The enzyme Histamine N-methyl transferase catalyzes N-methylation which means that it inactivates and degrades histamine. Since histamine is a key ingredient in allergic asthma, it works as a mediator with information from the mast cells. In allergic asthma, IgE antibodies will be released in response to the antibody-mediated cell degranulation. The gene coding for the HNMT protein is stated to be a candidate for inherited asthma due to the human polymorphism and primary biotransformation of histamine in the bronchial epithelium. Histamine N-methyltransferase is one of two enzymes involved in the metabolism of histamine, the second being diamine oxidase. Low levels of HNMT expression in numerous tissues points to an increased risk of asthma development. (Yan, 2000), (Yamauchi, 2019)

Expression system

The part was expressed with a T7-RNA-polymerase promoter (BBa_I719005), as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in Escherichia coli (E. coli) (BBa_K1758106).

Figure 2. Benchling screenshot of the expression system. The T7-RNA-polymerase promoter followed by a T7 g10 leader sequence increases efficiency of translation initiation. In order to increase the translation rate Poly A spacer, G10-L and AT-rich spacers were introduced into the sequence. His tag was added to the sequence in order to perform his tag purification later on in the experiments. TEV site was used in the plasmid construct for removing the his tag after the purification. BBa_B0015 was used as a double terminator.

Results

The transformation was partially successful. Small slow-growing colonies of E. coli (BL21) cells appeared after one night incubation at 37 °C (unusual for phenotype for BL21). When we further tried to purify any plasmid present in these cells, an agarose gel showed no presence of the plasmid. This indicates that the HNMT enzyme may be toxic to BL21 cells or that the antibiotic concentration used was slightly too low.