Histamine Dehydrogenase

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Introduction

Histamine is an essential biogenic amine in prokaryotes and tissues of animals and plants. In humans, histamine acts as a neurotransmitter, mediates allergic responses, plays a role in cell proliferation and in signaling the release of gastric acid into the stomach. Histamine receptors are targets for drugs that treat allergies and stomach acidity, but little structural information is available about these proteins. Although histamine oxidase from Arthrobacter globiformis has been used as a histamine sensor, it was later found that the enzyme is more sensitive to dopamine and tyramine than to histamine.

Histamine dehydrogenase (HADH) can be isolated from cultures of Nocardiodes simplex grown on histamine as the sole nitrogen source. The enzyme is a homodimer of a ∼76kDa subunit responsible for catalyzing the oxidative deamination of histamine to give imidazole acetaldehyde (Scheme 1), where the enzyme exhibits remarkable selectivity for histamine, thus showing great potential for use in biosensors. HADH was originally classified as a quinone-containing amine dehydrogenase. Later studies revealed that HADH is a homologue of TMADH, and dimethylamine dehydrogenase (DMADH) from M. methylotrophus shares 40% sequence identity and 56% similarity with both proteins.

Scheme 1 (Reed, T.; et al. 2010)

The uncommon 6-S-cysteinyl-FMN, or 6-S-Cys-FMN and [4Fe-4S] act as cofactors in this small protein family. The oxidation of histamine by HADH leaves the flavin in its reduced form, 6-S-Cys-FMN_red, which is reoxidized by a stepwise electron transfer reaction through the [4Fe-4S] to either a mediator, such as phenazinemethosulfate or, in TMADH. If HADH or TMADH decreases under single turnover conditions, a disproportionation reaction occurs between 6-S-Cys-FMN_red and [4Fe-4S]²⁺ to produce a semiquinone, 6-S-Cys-FMN_sq, and [ 4Fe-4S]⁺ (Scheme 2). In TMADH, the two spins form a triplet, which is detected by EPR as a strong half-field signal. Under steady-state conditions at high substrate concentrations, the 6-S-Cys-FMN_sq is stabilized by the binding of a substrate cation and can no longer be oxidized by [4Fe-4S], resulting in substrate inhibition.

Scheme 2 (Reed, T.; et al. 2010)

The Overall Structure of HADH

HADH is a homodimer containing two molecules per asymmetric unit, and its overall structure is very similar to that of the native TMADH. Full-length HADH contains 690 residues. Each molecule contains 6-S-Cys-FMN and [4Fe-4S] as redox active cofactors, and a single ADP is also present per molecule. Each subunit consists of a large domain (residues 7-385), a medium domain (residues 386-491 and 622-690) and a small domain (residues 492-621). The large domain contains an N-terminal triose-phosphate isomerase barrel, which is the most common tertiary fold in protein crystal structures. The 6-S-Cys-FMN is located at the opening of the barrel, surrounded by alpha-helices and large excursions at the end of the barrel's beta-strand to cover and bury the 6-S-Cys-FMN. The [4Fe-4S] cluster is coordinated to four Cys residues (Cys³⁴⁸, Cys³⁵¹, Cys³⁵⁴ and Cys³⁶⁶). As predicted from the sequence alignment, each protein molecule binds to one ADP molecule, located in the medium domain. ADP is exposed on the surface, close to the interface of the small and medium domains, but is not covalently linked.

The electron density of the 6-S-Cys-FMN is very clear, consistent with the fully flavinylated enzyme. The average occupancy of FMN in TMADH (PDB code 1DJN) is estimated to be 0.55. According to the electron density, it is speculated that the isoalloxazine ring of 6-S-Cys-FMN in recombinant HADH is not planar, but is in a "butterfly bend" conformation centered on the N5 and N10 positions, as shown in recombinant TMADH. Although the factors responsible for this butterfly bend are unknown, the cysteinyl crosslink formation has been shown to not cause the bend.

Figure 1. overview of recombinant HADH structure (Reed, T.; et al. 2010)