Heme And Heme Roman Branch Synthesis Key Enzyme In Structural Biology Research

1. The functional analyses of gene fragments encoding maize uroporphyrinogen methyltransferaseS-adenosylmethionine-dependent uroporphyrinogen methyltransferase (SUMT) is a key regulatory enzyme in the biosynthetic pathway of siroheme, heme d1, F₄₃₀ factor and vitamin B₁₂. In high plant, SUMT is responsible for the regulatory biosynthesis of siroheme. SUMT converts uroporphyrinogen III into precorrin-2 at C2 and C7 methylation. Overexpression of the gene encoding SUMT in Escherchia coli results in the celluar accumulation of the sirohydrochlorin, and an overmethylated compound trimethylpyrrocorphin. Both compounds emit strong red fluorescence under the UV light illumination. This character is applied as the assay of SUMT activity in vivo.The different gene fragments from maize cDNA were cut using the different restrictive endonucleases and inserted into different expression plasmids respectively. The individual encoding products of different gene fragments are maize SUMT, the SUMT precursor representing the signal peptide fused with the SUMT, the shorten enzyme lack of the S-adenosylmethionine binding motif, and the truncated enzyme deleted C-terminal 52 amino acid residues from the SUMT. The expressed maize SUMT and truncated enzyme are fused with His6-tag at N-terminus respectively. All the recombinant plasmids were transformed into E. coli JM109 correspondingly. The results showed that the individual E. coli colonies harboring the recombinant plasmids with the second and third gene fragments were not fluorescent. Further analyses identified the two gene products were expressed as inclusion bodies respectively, suggesting the chloroplast transit peptide or the S-adenosylmethionine binding motif were involved in affecting the protein folding in the heterogeneously expressed in E. coli. The red fluorescence were displayed in the cells containing the recombinant plasmids that expressing the SUMT fused with a 13 amino acid residues, or his-tag, or matalose binding protein, suggesting these fusions did not affect the enzyme activity significantly in vivo. The truncated enzyme was expressed actively, denoting the C-terminal 52 amino acid residues were not obviously contributed to SUMT activity.The purity of the SUMT and the truncated enzyme were up to 90% by Ni-NTA affinity chromatography after they were expressed in E. coli S13009. The purified maize SUMT was a homodimer with a molecular weight of 34 kD, as shown by SDS-PAGE, 52 kD using the gel-filtration chromatography, and 79 kD by dynamic light scattering assay. The results showed that maize SUMT was a homodimer in solution. The SUMT and truncated enzyme were associated with the red pigments.The pigments extracted from the purified SUMT were identified as trimethylpyrrocorphin as the main ingredient by UV-visible spectra. The emission peak shift was observed in the pigment-bound SUMT by fluorescent spectra, probably resulted from the interference of the amino acid residues in maize SUMT. The association of the pigments with the SUMT slight altered the protein secondary structure, confirmed by circular dichroism assay. The possible function of association for the pigments was discussed.2. Crystal structure of uroporphyrinogen decarboxylase from Bacillus subtilisUroporphyrinogen decarboxylase (UROD) is a branchpoint enzyme in the biosynthesis of the tetropyrroles. It catalyzes decarboxylation of four acetate groups of uroporphyrinogen III or the isomer I to yield coproporphyrinogen III or the isomer I, leading to heme and chlorophyll biosynthesis. UROD is a special type of non-oxidative decarboxylase since no cofactor required for catalysis.The hemE gene encoding UROD from Bacillus subtilis (bsUROD) was amplified by PCR using the genome DNA as the template. The sequencing analysis confirmed two base changes in the amplified gene product, which led to the replacement of Ilel56 and Glul98 by Thrl56 and Lysl98 in the expressing product correspondingly. The N-terminal hexahistidine-tagged bsUROD was purified up to 90% by Ni-NTA column. The specific activity of bsUROD is about 560U/mg protein.SDS-PAGE showed that monomer size of bsUROD is about 40 kDa. The monodispersive DLS data were consistent with a homodimer of about 81 kDa in solution.The hexahistidine-tagged bsUROD was crystallized by the hanging-drop method at 20℃. The diffraction data were collected from one crystal at 2.3 A resolution. The structure of bsUROD was solved by molecular replacement method using the human UROD as the model. The crystal of bsUROD had four molecules in the crystallographic asymmetric unit of P₁ cell. The cell dimensions were a=58.612A, b=80.410A, c=90.940 A,α=68.681°,β=89.638°,γ=80.817°. The final refinements of bsUROD resulted in an R factor and Rf_free factor of 19.7% and 25.1%, respectively. The RMSD from the ideal values of bond lengths and bond angles were 0.012 A and 1.290°. The atomic coordinates and diffraction data have been deposited to Protein Data Bank (http://www.pdb.org/) with an entry code of 2INF.The structure of bsUROD monomer included amino acid residues 6-88 and 101-349. The bsUROD monomer comprised a single domain which consists of a (β/α)₈ barrel fold. The active site structure in bsUROD monomer was a deep cleft. Aligning with coproporphyrin III in human UROD complex structure with bsUROD, eighteen amino acid residues in bsUROD was contacted with coproporphyrin III ligand at least one atom within 4.0 A. The residues were divided into three categories according to their function in substrate recognition and decarboxylation. The first category contained Asp78. The second category included a number of polar residues in the active cleft. The third category encompassed several hydrophobic amino acid residues. Among eighteen amino acid residues in bsUROD, five polar residues (Arg29, Arg33, Asp78, Tyr154, and His322), and three hydrophobic residues (Ile79, Phe144 and Phe207) were invariant in 30 organisms. The possible functions of these residues were discussed.Structural comparison of bsUROD with eukaryotic URODs revealed the variation of two loops, both of which possibly affected the binding of substrate and release of product. Since UROD and SUMT shared the common substrate, the essential amino acid residues for binding pyrrole NH group for uroporphyrinogen III in SUMT was speculated to be the same as that in bsUROD.A new catalyzing mechanism for bsUROD was proposed. Arg29 in bsUROD was favored to the binding of acetate group of substrate. Phe144 and/or Phe207 aromatic hydrogen might capture the CO₂ molecule. 3. Overexpression, purification and crystallization of some enzymes involved in the biosynthesis of heme, siroheme, and NAD.Heme and siroheme are the multi-functional groups in the cells. The function and structure of enzymes involved in the biosynthesis of heme and siroheme have been studied in recent years. 3-Hydroxyanthranilic acid 3, 4-dioxygenase (HAO) is a key regulatory enzyme in NAD biosynthesis.Except UROD, five enzymes involved in heme and siroheme synthesis from B. subtilis were overexpressed and purified. Only two enzymes were crystallized and their diffraction data were collected respectively. Two enzymes responsible for the terminal pathway of heme biosynthesis and HAO from Saccharomyces cerevisiae were also overexpressed and purified. Only HAO was crystallized and its structure has been solved.Based on the results of purification and crystal growth for some enzymes responsible for heme, siroheme and NAD biosynthesis, we briefly discussed the application of bioinformatics in crystal growth and the effect of histidine-tag on the protein purification, enzyme activity and the X-ray diffracting resolution of crystal.