Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps.

Hydroxymethylbilane synthase (HMBS), which is involved in the heme biosynthesis pathway, has a dipyrromethane cofactor and combines four porphobilinogen (PBG) molecules to form a linear tetrapyrrole, hydroxymethylbilane. Enzyme kinetic study of human HMBS using a PBG-derivative, 2-iodoporphobilinogen (2-I-PBG), exhibited noncompetitive inhibition with the inhibition constant being 5.4 ± 0.3 µM. To elucidate the reaction mechanism of HMBS in detail, crystal structure analysis of 2-I-PBG-bound holo-HMBS and its reaction intermediate possessing two PBG molecules (ES2), and inhibitor-free ES2 was performed at 2.40, 2.31, and 1.79 Šresolution, respectively. Their overall structures are similar to that of inhibitor-free holo-HMBS, and the differences are limited near the active site. In both 2-I-PBG-bound structures, 2-I-PBG is located near the terminus of the cofactor or the tetrapyrrole chain. The propionate group of 2-I-PBG interacts with the side chain of Arg173, and its acetate group is associated with the side chains of Arg26 and Ser28. Furthermore, the aminomethyl group and pyrrole nitrogen of 2-I-PBG form hydrogen bonds with the side chains of Gln34 and Asp99, respectively. These amino acid residues form a single substrate-binding site, where each of the four PBG molecules covalently binds to the cofactor (or oligopyrrole chain) consecutively, ultimately forming a hexapyrrole chain. Molecular dynamics simulation of the ES2 intermediate suggested that the thermal fluctuation of the lid and cofactor-binding loops causes substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. Finally, the hexapyrrole chain is hydrolyzed self-catalytically to produce hydroxymethylbilane.

Specificity of Fur binding to the oxidative stress response gene promoter in the facultative anaerobic archaeon Thermoplasma volcanium.

The genome of the facultative anaerobic thermoacidophilic archaeon Thermoplasma volcanium contains the open-reading frames (ORFs) tvsod and tvogg, which are predicted to encode a putative superoxide dismutase and an 8-oxoguanine DNA glycosylase, respectively. Tvsod is immediately upstream of tvogg, and these two ORFs are aligned in a head-to-tail manner in a single operon. A previous study showed that T. volcanium contains an ORF (TVN0292) encoding the ferric uptake regulator (Fur) and that the T. volcanium Fur protein (TvFur) binds to its own promoter in a metal-dependent manner in vitro. Here, we demonstrated that TvFur also binds to the tvsod-tvogg promoter and determined the TvFur-binding sequences in the tvsod-tvogg promoter by DNaseI footprinting analysis. These results suggest that Fur is required for resistance against reactive oxygen species in this facultative anaerobic archaeon.

Transcriptional factor fur from Thermoplasma volcanium binds its own promoter DNA in a divalent cation-dependent manner.

Because archaea possess many respiratory enzymes or radical scavengers with catalytic domains that contain iron, the expression of the genes encoding these enzymes might be regulated by iron acquisition. The genome of an archaeon, Thermoplasma volcanium contains a gene that encodes Fur (TVN0292). The fur gene of T. volcanium was amplified by PCR, and cloned into plasmid pET28a. TvFur (T. volcanium Fur protein) was expressed in E. coli cells and then purified. EMSA revealed that TvFur binds to its own promoter DNA. The binding to its own promoter was in an Mn(2+)-, Zn(2+)-, and Ni(2+)-dependent manner. DNase I footprinting analysis revealed that the binding sequence of tvfur promoter was 5'-G TTATTAT G TTTATAT A TTAATTA G-3'. An analysis utilizing oligonucleotides in TvFur-binding sequences revealed that TvFur binds to the TATA-box or regions in the vicinity of the TATA-box in the promoter. These results indicated that TvFur regulates transcription depending on the availability of environmental divalent cations.

Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli.

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase. Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity.

Characterization of rat heme oxygenase-3 gene. Implication of processed pseudogenes derived from heme oxygenase-2 gene.

Heme oxygenase (HO) is an enzyme responsible for the physiological degradation of heme to produce iron, CO and biliverdin. The released iron is recycled and represents the major source of this metal in heme homeostasis. A putative role as messenger in a signaling pathway is suggested for CO. Biliverdin, together with bilirubin, may function as an antioxidant. Thus far, three isoforms of HO, HO-1, HO-2 and HO-3 have been described. While HO-1 and HO-2 have been extensively investigated, HO-3 is still an elusive and poorly understood isoform. In this study, we examined the structure of the rat HO-3 gene with genomic PCR. However, we failed to isolate the reported HO-3 gene but, instead, found two HO-3-related genes, tentatively named HO-3a and HO-3b, whose sequences differed slightly from each other. Neither gene had any introns and consisted only of exon 2 through 5 of the HO-2 gene, though their sequences were not completely identical with that of HO-2. A stop codon was introduced within the coding regions of these genes due to frame-shift. The nucleotide sequence of their 5'-upstream region largely agreed with long interspersed nuclear element 3. No HO-3-related mRNAs were amplified by RT-PCR, and no HO-3-related proteins were detected in tissues by Western blot analysis. Our results suggested that there are no functional HO-3 genes in rat and that the HO-3a and HO-3b genes are processed pseudogenes derived from HO-2 transcripts.

Purification and characterization of a soluble form of rat liver NADPH-cytochrome P-450 reductase highly expressed in Escherichia coli.

A recombinant cDNA of rat liver NADPH-cytochrome P-450 reductase (CPR), which lacks the N-terminal hydrophobic region, was amplified by PCR and cloned. The N-truncated cDNA named tCPR was ligated into a pBAce vector and expressed. The tCPR protein expressed in Escherichia coli was recovered into the soluble fraction of the cell lysate and purified to homogeneity by three sequential purification procedures; (I) anion-exchange chromatography on a DEAE-cellulose (DE-52) column, (II) affinity chromatography on 2('),5(')-ADP Sepharose 4B, and (III) chromatography on a hydroxyapatite column. The average yield was 47mg per liter of culture medium. The absorption spectrum of the purified tCPR protein was identical to that of a native full-length CPR purified from rat liver, indicating that tCPR also possesses one molecule each of FAD and FMN. The tCPR protein was able to reduce cytochrome c and was also able to assist heme degradation by a soluble form of rat heme oxygenase-1. However, it failed to support the O-deethylation of 7-ethoxycoumarin by cytochrome P-450 1A1, indicating that the presence of the N-terminal hydrophobic domain is necessary for CPR to interact with cytochrome P-450. Previously, to prepare a soluble form of CPR, full-length CPR was treated with proteinases that selectively removed the N-terminal domain. With the expression system established in this study, however, the soluble and biologically active tCPR protein can be readily prepared in large amounts. This expression system will be useful for mechanistic as well as structural studies of CPR.

The reactivity of alpha-hydroxyhaem and verdohaem bound to haem oxygenase-1 to dioxygen and sodium dithionite.

Recently we have shown that ferric alpha-hydroxyhaem bound to haem oxygenase-1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196-18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric alpha-hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous alpha-hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203-213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. alpha-hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of alpha-hydroxyhaem with haem oxygenase may be small and a substantial amount of free alpha-hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air-sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH-cytochrome P450 reductase.

Crystal structure of rat heme oxygenase-1 in complex with heme bound to azide. Implication for regiospecific hydroxylation of heme at the alpha-meso carbon.

Heme oxygenase (HO) catalyzes physiological heme degradation consisting of three sequential oxidation steps that use dioxygen molecules and reducing equivalents. We determined the crystal structure of rat HO-1 in complex with heme and azide (HO-heme-N(3)(-)) at 1.9-A resolution. The azide, whose terminal nitrogen atom is coordinated to the ferric heme iron, is situated nearly parallel to the heme plane, and its other end is directed toward the alpha-meso position of the heme. Based on resonance Raman spectroscopic analysis of HO-heme bound to dioxygen, this parallel coordination mode suggests that the azide is an analog of dioxygen. The azide is surrounded by residues of the distal F-helix with only the direction to the alpha-meso carbon being open. This indicates that regiospecific oxygenation of the heme is primarily caused by the steric constraint between the dioxygen bound to heme and the F-helix. The azide interacts with Asp-140, Arg-136, and Thr-135 through a hydrogen bond network involving five water molecules on the distal side of the heme. This network, also present in HO-heme, may function in dioxygen activation in the first hydroxylation step. From the orientation of azide in HO-heme-N(3)(-), the dioxygen or hydroperoxide bound to HO-heme, the active oxygen species of the first reaction, is inferred to have a similar orientation suitable for a direct attack on the alpha-meso carbon.

Crystal structure of rat apo-heme oxygenase-1 (HO-1): mechanism of heme binding in HO-1 inferred from structural comparison of the apo and heme complex forms.

Heme oxygenase (HO) catalyzes the oxidative cleavage of heme to biliverdin by utilizing O(2) and NADPH. HO (apoHO) was crystallized as twinned P3(2) with three molecules per asymmetric unit, and its crystal structure was determined at 2.55 A resolution. Structural comparison of apoHO and its complex with heme (HO-heme) showed three distinct differences. First, the A helix of the eight alpha-helices (A-H) in HO-heme, which includes the proximal ligand of heme (His25), is invisible in apoHO. In addition, the B helix, a portion of which builds the heme pocket, is shifted toward the heme pocket in apoHO. Second, Gln38 is shifted toward the position where the alpha-meso carbon of heme is located in HO-heme. Nepsilon of Gln38 is hydrogen-bonded to the carbonyl group of Glu29 located at the C-terminal side of the A helix in HO-heme, indicative that this hydrogen bond restrains the angle between the A and B helices in HO-heme. Third, the amide group of Gly143 in the F helix is directed outward from the heme pocket in apoHO, whereas it is directed toward the distal ligand of heme in HO-heme. This means that the F helix around Gly143 must change its conformation to accommodate heme binding. The apoHO structure has the characteristic that the helix on one side of the heme pocket fluctuates, whereas the rest of the structure is similar to that of HO-heme, as observed in such hemoproteins as myoglobin and cytochromes b(5) and b(562). These structural features of apoHO suggest that the orientation of the proximal helix and the position of His25 are fixed upon heme binding.