Crystal Structure of Uroporphyrinogen Decarboxylase from Bacillus subtilis

Abstract

Uroporphyrinogen decarboxylase (UROD) is a branch point enzyme in the biosynthesis of the tetrapyrroles. It catalyzes the decarboxylation of four acetate groups of uroporphyrinogen III to yield coproporphyrinogen III, leading to heme and chlorophyll biosynthesis. UROD is a special type of nonoxidative decarboxylase, since no cofactor is essential for catalysis. In this work, the first crystal structure of a bacterial UROD, Bacillus subtilis UROD (UROD Bs ), has been determined at a 2.3 Å resolution. The biological unit of UROD Bs was determined by dynamic light scattering measurements to be a homodimer in solution. There are four molecules in the crystallographic asymmetric unit, corresponding to two homodimers. Structural comparison of UROD Bs with eukaryotic URODs reveals a variation of two loops, which possibly affect the binding of substrates and release of products. Structural comparison with the human UROD-coproporphyrinogen III complex discloses a similar active cleft, with five invariant polar residues (Arg29, Arg33, Asp78, Tyr154, and His322) and three invariant hydrophobic residues (Ile79, Phe144, and Phe207), in UROD Bs . Among them, Asp78 may interact with the pyrrole NH groups of the substrate, and Arg29 is a candidate for positioning the acetate groups of the substrate. Both residues may also play catalytic roles.Tetrapyrroles, including heme, chlorophyll, siroheme, and vitamin B 12 , are a family of structurally related cofactors and prosthetic groups with different central metal ion insertions. The common pathways in tetrapyrrole biosynthesis start from the first precursor 5-aminolevulinate. Two molecules of this compound are condensed to generate porphobilinogen (PBG). Four molecules of PBG are further condensed by PBG deaminase (PBGD) to generate hydroxymethylbilane, which is subsequently cyclized to generate either uroporphyrinogen III (urogen III), by urogen III synthase with inversion of the D pyrrole ring, or isomer urogen I, by nonenzyme catalysis. urogen III is the last common precursor of all tetrapyrroles in living cells, at which major branching of the biosynthetic pathways happens. Bismethylation of urogen III by urogen methyltransferase (UPMT) generates procorrin-2, an intermediate in the biosynthesis of siroheme and vitamin B 12 .

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

“…An X-ray crystallographic structure at 2.3 Åo fB. subtilis UroD was reported in 2007 (198), but there have been no follow-up studies. The Protein Data Bank (PDB) contains a 1.95-Å structure (accession number 4WSH) of a "probable" UroD enzyme from P. aeruginosa as well as a 1.9-Å structure (PDB accession number 2EJA) from Aquifex aeolicus, a 2.8-Å structure (accession number 3CYV) from Shigella flexneri, and a 1.6-Å structure (accession number 4EXQ) from Burkholderia thailandensis.…”

HemQ: An iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria

“…Among bacteria hemE is present in those organisms that are known to synthesize protoporphyrin IX, often in an operon or cluster with the genes encoding other enzymes of a 3-step protoheme IX biosynthetic module. The crystal structure of HemE has been obtained and shows that the protein is a homodimer with an active site in each monomer (32, 35). The enzyme structure is highly conserved between human and Bacillus subtilis , although the bacterial enzyme possesses two additional small loops.…”

Structure and function of enzymes in heme biosynthesis

“…The enzymes UROD, CPO and CPDH, PPO, and FC were subsequently purified from various eukaryotic and prokaryotic sources and the corresponding genes were cloned and sequenced 120–122. Crystal structures are available for human UROD in its apo and COPROGEN bound forms, as well as for Nicotiana tabacum and Bacillus subtilis UROD 123–126. CPO was crystallized and the structures solved for the yeast and human enzymes 127, 128.…”