Juha P. Kallio

Hydroxymethylbilane synthase (HMBS), involved in haem biosynthesis, catalyses the head‐to‐tail coupling of four porphobilinogens (PBGs) via a dipyrromethane (DPM) cofactor. DPM is composed of two PBGs, and a hexapyrrole is built before the tetrapyrrolic 1‐hydroxymethylbilane product is released. During this elongation, stable enzyme (E) intermediates are formed from the holoenzyme, with additional PBG substrates (S): ES, ES 2 , ES 3 and ES 4 . Native PAGE and mass spectrometry of the acute intermittent porphyria (AIP)‐associated HMBS variant p.Arg167Gln demonstrated an increased amount of ES 3 . Kinetic parameters indicated catalytic dysfunction, however, the product release was not entirely prevented. Isolation and crystal structure analysis of the ES 3 intermediate (PDB: 8PND ) showed that a pentapyrrole was fully retained within the active site, revealing that polypyrrole elongation proceeds within the active site via a third interaction site, intermediate pyrrole site 3 (IPS3). The AIP‐associated HMBS variant p.Arg195Cys, located on the opposite side to p.Arg167Gln in the active site, accumulated the ES 4 intermediate in the presence of excess PBG, implying that product hydrolysis was obstructed. Arg167 is thus involved in all elongation steps and is a determinant for the rate of enzyme catalysis, whereas Arg195 is important for releasing the product. Moreover, by substituting residues in the vicinity of IPS3, our results indicate that a fully retained hexapyrrole could be hydrolysed in a novel site in proximity of the IPS3.

Hydroxymethylbilane synthase (HMBS) is the third enzyme involved in haem biosynthesis, in which it catalyses the formation of tetrapyrrole 1‐hydroxymethylbilane (HMB). In this process, HMBS binds four consecutive substrate molecules, creating the enzyme‐intermediate complexes ES, ES 2 , ES 3 and ES 4 . Pathogenic variants in the HMBS gene are associated with the dominantly inherited disorder acute intermittent porphyria. In this study, we have characterised the p.R26H variant to shed light on the role of Arg26 in the elongation mechanism of HMBS and to provide insights into its effect on the enzyme. With selected biophysical methods, we have been able to show that p.R26H forms a single enzyme‐intermediate complex in the ES 2 ‐state. We were also able to demonstrate that the p.R26H variant results in an inactive enzyme, which is unable to produce the HMB product.