Journal Article

PUBDB-2024-00254

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png One ring closer to a closure: the crystal structure of the ES$_3$ hydroxymethylbilane synthase intermediate

Bustad, H. J. ; Christie, M. S. ; Laitaoja, M. ; Aarsand, A. K. ; Martinez, A. ; Jänis, J. ; Kallio, J. P. (Corresponding author)Extern*EMBL*

2023
Wiley-Blackwell Oxford [u.a.]

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Please use a persistent id in citations: doi:10.1111/febs.16982  doi:10.3204/PUBDB-2024-00254

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, 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.

Note: online first

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Contributing Institute(s):

1. DOOR-User (DOOR ; HAS-User)

Research Program(s):

1. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)

Experiment(s):

1. PETRA Beamline P11 (PETRA III)

Appears in the scientific report 2023

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