Dataset Information

Substrate Specificity of the Flavoenzyme BhaC₁ That Converts a C-Terminal Trp to a Hydroxyquinone.

ABSTRACT: The preparation of protein-protein, protein-peptide, and protein-small molecule conjugates is important for a variety of applications, such as vaccine production, immunotherapies, preparation of antibody-drug conjugates, and targeted delivery of therapeutics. To achieve site-selective conjugation, selective chemical or enzymatic functionalization of proteins is required. We have recently reported biosynthetic pathways in which small, catalytic scaffold peptides are utilized for the generation of amino acid-derived natural products called pearlins. In these systems, peptide amino-acyl tRNA ligases (PEARLs) append amino acids to the C-terminus of a scaffold peptide, and tailoring enzymes encoded in the biosynthetic gene clusters modify the PEARL-appended amino acid to generate a variety of natural products. Herein, we investigate the substrate selectivity of one such tailoring enzyme, BhaC₁, that participates in pyrroloiminoquinone biosynthesis. BhaC₁ converts the indole of a C-terminal tryptophan into an o-hydroxy-p-quinone, a promising moiety for site-selective bioconjugation. Our studies demonstrate that BhaC₁ requires a 20-amino acid peptide for substrate recognition. When this peptide was appended at the C-terminus of proteins, the C-terminal Trp was modified by BhaC₁. The enzyme is sufficiently selective that only small changes to the sequence of the peptide are tolerated. An AlphaFold model for substrate recognition explains the selectivity of the enzyme, which may be used to install a reactive handle onto the C-terminus of proteins.

SUBMITTER: Daniels PN

PROVIDER: S-EPMC9850906 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Substrate Specificity of the Flavoenzyme BhaC<sub>1</sub> That Converts a C-Terminal Trp to a Hydroxyquinone.

Daniels Page N PN van der Donk Wilfred A WA

Biochemistry 20220525 2

The preparation of protein-protein, protein-peptide, and protein-small molecule conjugates is important for a variety of applications, such as vaccine production, immunotherapies, preparation of antibody-drug conjugates, and targeted delivery of therapeutics. To achieve site-selective conjugation, selective chemical or enzymatic functionalization of proteins is required. We have recently reported biosynthetic pathways in which small, catalytic scaffold peptides are utilized for the generation of ...[more]

PMID: 35613706

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Project description:Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate=MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions that should help rationalizing substrate-specificity and lay the ground for inhibitor design.

Dataset Information

Substrate Specificity of the Flavoenzyme BhaC₁ That Converts a C-Terminal Trp to a Hydroxyquinone.

Publications

Substrate Specificity of the Flavoenzyme BhaC<sub>1</sub> That Converts a C-Terminal Trp to a Hydroxyquinone.

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Dataset Information

Substrate Specificity of the Flavoenzyme BhaC1 That Converts a C-Terminal Trp to a Hydroxyquinone.

Publications

Substrate Specificity of the Flavoenzyme BhaC<sub>1</sub> That Converts a C-Terminal Trp to a Hydroxyquinone.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Substrate Specificity of the Flavoenzyme BhaC₁ That Converts a C-Terminal Trp to a Hydroxyquinone.