Crystal structure of histamine dehydrogenase from Nocardioides simplex.

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.

Crystal structure of prolyl 4-hydroxylase from Bacillus anthracis.

Prolyl 4-hydroxylases (P4H) catalyze the post-translational hydroxylation of proline residues and play a role in collagen production, hypoxia response, and cell wall development. P4Hs belong to the group of Fe(II)/alphaKG oxygenases and require Fe(II), alpha-ketoglutarate (alphaKG), and O(2) for activity. We report the 1.40 A structure of a P4H from Bacillus anthracis, the causative agent of anthrax, whose immunodominant exosporium protein BclA contains collagen-like repeat sequences. The structure reveals the double-stranded beta-helix core fold characteristic of Fe(II)/alphaKG oxygenases. This fold positions Fe-binding and alphaKG-binding residues in what is expected to be catalytically competent orientations and is consistent with proline peptide substrate binding at the active site mouth. Comparisons of the anthrax P4H structure with Cr P4H-1 structures reveal similarities in a peptide surface groove. However, sequence and structural comparisons suggest differences in conformation of adjacent loops may change the interaction with peptide substrates. These differences may be the basis of a substantial disparity between the K(M) values for the Cr P4H-1 compared to the anthrax and human P4H enzymes. Additionally, while previous structures of P4H enzymes are monomers, B. anthracis P4H forms an alpha(2) homodimer and suggests residues important for interactions between the alpha(2) subunits of alpha(2)beta(2) human collagen P4H. Thus, the anthrax P4H structure provides insight into the structure and function of the alpha-subunit of human P4H, which may aid in the development of selective inhibitors of the human P4H enzyme involved in fibrotic disease.

Expression, purification, crystallization and preliminary X-ray studies of histamine dehydrogenase from Nocardioides simplex.

Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 A resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 A.

Expression, purification, crystallization and preliminary X-ray studies of a prolyl-4-hydroxylase protein from Bacillus anthracis.

Collagen prolyl-4-hydroxylase (C-P4H) catalyzes the hydroxylation of specific proline residues in procollagen, which is an essential step in collagen biosynthesis. A new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human has recently been characterized. The structure of anthrax-P4H could provide important insight into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in Escherichia coli have been collected to 1.4 A resolution.

The P450cam G248E mutant covalently binds its prosthetic heme group.

Previous studies on mammalian peroxidases and cytochrome P450 family 4 enzymes have shown that a carboxylic group positioned close to a methyl group of the prosthetic heme is required for the formation of a covalent link between a protein carboxylic acid side chain and the heme. To determine whether there are additional requirements for covalent bond formation in the P450 enzymes, a glutamic acid or an aspartic acid has been introduced into P450(cam) close to the heme 5-methyl group. Spectroscopic and kinetic studies of the resulting G248E and G248D mutants suggest that the carboxylate group coordinates with the heme iron atom, as reported for a comparable P450(BM3) mutant [Girvan, H. M., Marshall, K. R., Lawson, R. J., Leys, D., Joyce, M. G., Clarkson, J., Smith, W. E., Cheesman, M. R., and Munro, A. W. (2004) J. Biol. Chem. 279, 23274-23286]. The two P450(cam) mutants have low catalytic activity, but in contrast to the P450(BM3) mutant, incubation of the G248E (but not G248D) mutant with camphor, putidaredoxin, putidaredoxin reductase, and NADH results in partial covalent binding of the heme to the protein. No covalent attachment is observed in the absence of camphor or any of the other reaction components. Pronase digestion of the G248E P450(cam) mutant after covalent attachment of the heme releases 5-hydroxyheme, establishing that the heme is covalently attached through its 5-methyl group as predicted by in silico modeling. The results establish that a properly positioned carboxyl group is the sole requirement for autocatalytic formation of a heme-protein link in P450 enzymes, but also show that efficient covalent binding requires placement of the carboxyl close to the methyl but in a manner that prevents strong coordination to the iron atom.

Cloning and characterization of histamine dehydrogenase from Nocardioides simplex.

Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here, the hdh gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6+/-0.3 mol Fe and 3.7+/-0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histamine reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow substrate specificity, oxidizing only histamine (K(m)=31+/-11 microM, k(cat)/K(m)=2.1 (+/-0.4)x10(5)M(-1)s(-1)), agmatine (K(m)=37+/-6 microM, k(cat)/K(m)=6.0 (+/-0.6)x10(4)M(-1)s(-1)), and putrescine (K(m)=1280+/-240 microM, k(cat)/K(m)=1500+/-200 M(-1)s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K(m) (histamine) and the measurement of a substrate deuterium isotope effect, (D)(k(cat)/K(m) (histamine))=7.0+/-1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat))=1.3+/-0.2.

Two new terpyridine dimanganese complexes: a manganese(III,III) complex with a single unsupported oxo bridge and a manganese(III,IV) complex with a dioxo bridge. Synthesis, structure, and redox properties.

Two new terpyridine dimanganese oxo complexes [Mn(2)(III,IV)(mu-O)(2)(terpy)(2)(CF(3)CO(2))(2)](+) (3) and [Mn(2)(III,III)(mu-O)(terpy)(2)(CF(3)CO(2))(4)] (4) (terpy = 2,2':6,2' '-terpyridine) have been synthesized and their X-ray structures determined. In contrast to the corresponding mixed-valent aqua complex [Mn(2)(III,IV)(mu-O)(2)(terpy)(2)(H(2)O)(2)](3+) (1), the two Mn atoms in 3 are not crystallographically equivalent. The neutral binuclear monooxo manganese(III,III) complex 4 exhibits two crystallographic forms having cis and trans configurations. In the cis complex, the two CF(3)CO(2)(-) ligands on each manganese adopt a cis geometry to each other; one CF(3)CO(2)(-) is trans to the oxygen of the oxo bridge while the second is cis. In the trans complex, the two coordinated CF(3)CO(2)(-) have a trans geometry to each other and are cis to the oxo bridge. The electrochemical behavior of 3 in organic medium (CH(3)CN) shows that this complex could be oxidized into its corresponding stable manganese(IV,IV) species while its reduced form manganese(III,III) is very unstable and leads by a disproportionation process to Mn(II) and Mn(IV) complexes. Complex 4 is only stable in the solid state, and it disproportionates spontaneously in CH(3)CN solution into the mixed-valent complex 3 and the mononuclear complex [Mn(II)(terpy)(2)](2+) (2), thereby preventing the observation of its electrochemical behavior.