8UW4

Crystal structure of hydroxyisourate hydrolase from Herbaspirillum seropedicae

Summary for 8UW4

• Entry DOI: 10.2210/pdb8uw4/pdb
• Descriptor: 5-hydroxyisourate hydrolase (2 entities in total)
• Functional Keywords: enzyme, hydrolase
• Biological source: Herbaspirillum seropedicae
• Total number of polymer chains: 8
• Total formula weight: 116980.26

Authors

Magalhaes, M.T.Q.,Pereira, H.M.,Bleicher, L. (deposition date: 2023-11-06, release date: 2024-01-31, Last modification date: 2025-02-12)

Primary citation

Lemos, R.P.,Rodrigues, J.T.,Portwood, G.,de Oliveira, L.C.,Gomes Dos Santos, P.H.,Costa, M.A.F.,Pereira, H.D.,Bleicher, L.,de Magalhaes, M.T.Q.
Evolution-based protein engineering: functional switching between transthyretins and 5-hydroxyisourate hydrolases.
J.Biomol.Struct.Dyn., :1-17, 2024

PubMed Abstract: Transthyretin (TTR) is a vertebrate-exclusive transport protein that plays a key role in binding and distributing thyroid hormones. However, its evolutionary origin lies in the duplication of the gene that encoding the enzyme 5-hydroxyisourate hydrolase (HIUase), which is involved in uric acid metabolism. Unlike TTR, HIUase is ubiquitous in both prokaryotes and eukaryotes, with the exception of hominids. Both HIUase and TTR subfamilies form homotetramers, possessing an internal charged cavity between the two dimer pairs. Based on their high degree of structural similarity, we hypothesized that specific substitutions would enable the interconversion between these protein functions. Using an evolution-based approach, we engineered two putative protein sequences, where correlated locally conserved positions from one subfamily representative sequence were substituted by the other, and vice versa. Applying computational modeling techniques, the best models were refined, validated, and their cavity volumes, three-dimensional geometries, propensity to aggregation and electrostatic potentials were analyzed. Molecular dynamics simulations were performed with the reference proteins and the engineered mutants in the bound and unbound states. We demonstrate that the volumes and geometries differ from one another, due to size and physicochemical differences between their ligands. The bound state mutant complexes are stable, and the enzymatic assay demonstrated active new enzymes. Our work suggests that the evolution-based protein engineering approach used has residue-specific resolution to identify locally conserved residues in the sequence of evolutionarily related proteins, such as HIUase and TTR.

PubMed: 39705024
DOI: 10.1080/07391102.2024.2440647

Experimental method

X-RAY DIFFRACTION (2.15 Å)