One ring closer to a closure: the crystal structure of the ES3 hydroxymethylbilane synthase intermediate

Abstract

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, ES₂ , ES₃ and ES₄ . Native PAGE and mass spectrometry of the acute intermittent porphyria (AIP)-associated HMBS variant p.Arg167Gln demonstrated an increased amount of ES₃ . Kinetic parameters indicated catalytic dysfunction, however, the product release was not entirely prevented. Isolation and crystal structure analysis of the ES₃ 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 ES₄ 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.

Keywords: acute intermittent porphyria; haem biosynthesis; hydroxymethylbilane synthase; porphobilinogen; pyrrole elongation.