Structure and function of enzymes in heme biosynthesis

Abstract

Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V-shaped glutamyl-tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the "Radical SAM enzyme" coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure-function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.

Figure 1

Heme biosynthesis. A: The first cyclic tetrapyrrole uroporphyrinogen III is formed from the precursor 5-aminolevulinic acid in three enzymatic steps via the intermediates porphobilinogen and pre-uroporphyrinogen. Depending on the organism, ALA is either synthesized by condensation of glycine with succinyl-CoA or from tRNA-bound glutamate via glutamate-1-semialdehyde. B: Uroporphyrinogen III is converted into heme in four consecutive enzymatic steps via the intermediates coproporphyrinogen III, protoporphyrinogen IX, and protoporphyrin IX. Structures of all heme biosynthesis enzymes have been determined with the exception of oxygen-independent PPO (n.d., structure not determined).

Figure 2

Active site architectures and catalytic steps of enzymes involved in ALA formation. A + B, ALAS; C + D, GluTR; E + F, GSAM. A: Active site of ALAS from R. capsulatus modeled from the glycine- and succinyl-CoA-bound structures showing the external aldimine between PLP and glycine in close proximity to the succinyl-CoA CS1 position. B: During the ALAS reaction cycle, the decarboxylation of the α-amino-β-keto adipate intermediate is promoted by His-mediated protonation. C: Active site of GluTR from M. kandleri with bound glutamycin and the catalytically essential Cys(Ser)48. D: During catalysis Cys48 nucleophilically attacks the activated α-carboxylate of the glutamyl-tRNA. E: Active site of GSAM from Synechococcus showing the inhibitor gabaculine covalently bound to the PLP cofactor resulting in the stable m-carboxyphenylpyridoxalamine phosphate (mCPP). F: The potential reaction intermediate 4,5-diaminovalerate covalently bound to PLP as an external aldimine.

Figure 3

Active site architectures and catalytic steps of enzymes involved in uroporphyrinogen III formation. A + B, PBGS; C + D, PBGD; E + F, UROS. A: Active site of P. aeruginosa PBGS with bound inhibitor 5-fluorolevulinic acid (5F-LA). B: At the beginning of the PBGS reaction cycle both ALA molecules are covalently bound to the enzyme via Schiff bases to conserved lysine residues. C: Active site of human PBGD showing the unique dipyrromethane cofactor (DPM) covalently bound to an invariant cysteine residue. D: The oligomerization of PBG molecules proceeds via deaminated azafulvene intermediates. E: Active site of T. thermophilus UROS with bound uroporphyrinogen III (UROGEN) showing a puckered “two-up, two-down” conformation of the reaction product. F: Dehydration of pre-uroporphyrinogen results in the first azafulvene intermediate which further reacts to form a spirocyclic pyrrolenine intermediate as indicated by the arrows.

Figure 4

Active site architectures and catalytic steps of enzymes involved in protoporphyrinogen IX formation. A + B, UROD; C + D, CPO; E + F, CPDH. A: Active site of human UROD with bound coproporphyrinogen III (COPROGEN) in a dome-shaped conformation showing the catalytically essential aspartate in hydrogen bonding distance to the pyrrole NH-groups. B: The aspartate residue stabilizes the protonated, positively charged reaction intermediate. C: Active site of human CPO with bound citrate showing several arginine residues possibly involved in substrate binding and the catalytically essential aspartate. D: During catalysis, a pyrrole peroxide anion is formed which further reacts via proton abstraction through the peroxide to form an intermediate containing an exocyclic double bond. E: Active site of E. coli CPDH showing the catalytically essential [4Fe-4S] cluster and the two bound SAM molecules. F: During the initial reaction steps the reduced iron-sulfur cluster transfers an electron to SAM, which is thereby cleaved into methionine and a 5′-deoxyadenosyl radical. This radical then abstracts a hydrogen atom from the substrate propionate side chain resulting in the formation of 5′-deoxyadenosine and a substrate radical. R = tetrapyrrole.

Figure 5

Active site architectures and catalytic steps of enzymes involved in the transformation of protoporphyrinogen IX into heme. A + B, PPO; C + D, FC. A: Active site of M. xanthus PPO with bound inhibitor acifluorfen (AF). B: PPO catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. C: Active site of human FC with bound protoporphyrin IX (PROTO). D: The insertion of ferrous iron into PROTO proceeds via a “sitting-atop” complex between the porphyrin and the metal ion.