Structural basis of pyrrole polymerization in human porphobilinogen deaminase

Abstract

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.

Systematically Analyzing the Pathogenic Variations for Acute Intermittent Porphyria

“…In order to understand the effect of these missense mutations on HMBS protein structure, we mapped those mutations to the crystal structure (PDB: 5M7F, Resolution: 2.78- Å ) (Pluta et al, 2018) to study how the mutations affect the structure of the protein and then evaluated the relationship between mutations and clinical symptoms. For the visualization of the three-dimensional structure of protein, we used a web tool ().…”

Acute Intermittent Porphyria: An Overview of Therapy Developments and Future Perspectives Focusing on Stabilisation of HMBS and Proteostasis Regulators

“…Despite recent development in the field of HMBS by the determination of the X-ray structures of the human wild-type holoenzyme and ES 2 intermediate state [ 75 , 76 ], the details of the enzyme elongation mechanism and product release are still unclear ( Figure 3 ). Knowing the detailed mechanism is crucial for the understanding of different clinical phenotypes in patients, and why some disease-related variants affect the enzyme activity without obvious indications of altering the holoenzyme structure.…”

“…In the search for, e.g., pharmacological chaperones, well-defined crystal structures are particularly important in docking-based in silico screenings to identify and characterize binders. When working with HMBS, an enzyme with a complex multi-step mechanism, we would benefit even more if structural and mechanistic details beyond ES 2 intermediate state formation were known [ 75 ].…”

“…There are several published structures of HMBS ( Table 1 ); however, until 2018, only structures in the holo-state had been published. The structure of HMBS in the ES 2 intermediate state demonstrated how specific areas of the enzyme, as well as the growing pyrrole chain, moved during the elongation [ 75 ], adding to the complexity of the enzyme mechanism. In particular, the loop covering the active site (residues 58–74) is thought to be important in the catalytic process.…”

“…This loop has been suggested by in silico analysis to move from a ‘closed’ to an ‘open’ conformation during the elongation process, in agreement with its relevant role in catalysis [ 207 ]. The structure of the loop cannot be defined in most of the crystal structures, probably because of its dynamic properties, and has only been detected in the ‘closed’ state in the crystal structure of A. thaliana HMBS (PDB 4HTG) [ 72 ], and more recently in the ‘open’ state of the structures of human wild-type (PDB 5M6R) and p.Arg173Trp mutant (PDB 7AAK) [ 75 , 76 ]. However, the function, and participation of this dynamic loop in the catalytic mechanism are still unclear.…”