Abstract
Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-A resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S](2+)) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr(60), Trp(264), and Trp(355) provide the framework for the "aromatic bowl" that serves as a trimethylamine-binding site in TMADH is comprised of Gln(65), Trp(267), and Asp(358), respectively, in HADH. The surface Tyr(442) that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.
Highlights
25782 JOURNAL OF BIOLOGICAL CHEMISTRY ach acidity, but there is very little structural information on the histamine-binding sites of these proteins
The oxidation of histamine by HADH leaves the flavin in its reduced form, 6-S-Cys-FMNred, which is reoxidized by a stepwise electron transfer reaction through the [4Fe-4S] to either a mediator, such as phenazinemethosulfate [6] or, in Trimethylamine dehydrogenase (TMADH), 5 The abbreviations used are: TMADH, trimethylamine dehydrogenase; HADH, histamine dehydrogenase; DMADH, dimethylamine dehydrogenase; ETF, electron transferring flavoprotein; human histamine receptors (HHRs), human histamine receptor; SeMet, selenomethionine; HNMT, histamine N-methyltransferase; histamine-binding proteins (HBPs), histamine-binding protein; PDB, Protein data bank
Sequence alignment suggests that the active sites of TMADH and HADH are very different, EPR shows that the magnetic interactions between the 6-S-Cys-FMN and [4Fe4S] differ between TMADH and HADH
Summary
An electron transferring flavoprotein (ETF) [10]. If either HADH or TMADH are reduced under single turnover conditions, a disproportionation reaction occurs between 6-S-CysFMNred and [4Fe-4S]2ϩ to produce a semiquinone, 6-S-CysFMNsq, and [4Fe-4S]ϩ (Scheme 2) [6]. The equivalent positions in HADH from an amino acid sequence alignment are Gln, Glu, Ala110, Trp264, and Asp364 [6] These differences suggest that the histamine-binding site of HADH is significantly different from other flavin-dependent amine metabolizing enzymes. HADH exhibits a significant primary kinetic deuterium isotope effect using ␣,␣,,-d4-histamine on kcat/Km (Ͼ7.0) that is absent in kcat [6] This suggests that C-H bond cleavage, and 6-S-Cys-FMN reduction, is rate-limiting at subsaturating concentrations of histamine but that the subsequent steps, possibly electron transfer, define kcat. This contrasts with TMADH, where the effect of using (CD3)3NHϩ can only be examined by stopped-flow methods, and there is no isotope effect in the steady state [19]. We report the first three-dimensional structure of a histamine dehydrogenase
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