One ring closer to a closure: the crystal structure of the ES<inf>3</inf> hydroxymethylbilane synthase intermediate
Bustad, Helene J.; Sæter, Marthe Christie; Laitaoja, Mikko; Aarsand, Aasne Karine; Martinez, Aurora; Jänis, Janne; Kallio, Juha Pekka
Journal article, Peer reviewed
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Date
2023Metadata
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- Department of Biomedicine [743]
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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.