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, BhaC 1 , that participates in pyrroloiminoquinone biosynthesis. BhaC 1 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 BhaC 1 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 BhaC 1 . 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.

Characterization of a Ruthenium(II) Complex in Singlet Oxygen-Mediated Photoelectrochemical Sensing

“…To the sample thus obtained was added 10% deuterium oxide and kept in suspension within the active volume of the coil by confining it with low-melting agarose. This procedure has been established to keep cells in suspension in bioreactors. , …”

“…This procedure has been established to keep cells in suspension in bioreactors. 21,22 Computational Details. The structural and spectroscopic properties of the ruthenium complex have been carried out within the density-functional theory (DFT) framework with the Gaussian 09 suite of programs 23,24 using the PBE0 exchange and correlation functional and the LANL2DZ basis set.…”

Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

“…Peptide amino-acyl tRNA ligases were used to attach the recognition sequence into proteins allowing the formation of an electrophilic o-hydroxy-pquinone handle and subsequent labelling of targeted proteins. [213] Trp C-mannosyltransferase (CMT) enzymes are involved in function, transport, and folding of secretory and transmem-brane proteins. [214] The C. elegans DPY-19 is a Trp CMT glycosylating enzyme.…”

“…This broad substrate flexibility was applied to maltose binding protein, resulting in a handle that can subsequently be used for bioconjugation. Peptide amino‐acyl tRNA ligases were used to attach the recognition sequence into proteins allowing the formation of an electrophilic o‐ hydroxy‐ p ‐ quinone handle and subsequent labelling of targeted proteins [213] …”

Unexpected Transformations during Pyrroloiminoquinone Biosynthesis

“…15 ■ RESULTS AND DISCUSSION Glycine Oxidase BhaG Oxidizes the Glycine-Quinone Adduct 1. To uncover the next biosynthetic step in the bha pathway, the peptide BhaA-Ala-Trp was heterologously coexpressed in with the previously characterized modifying enzymes (BhaC 1 and BhaB 5 ; Figure 1D) 4,16 as well as iterative inclusion of each of the remaining enzymes encoded in the bha cluster (Figure 1B). After peptide purification and desalting, the samples were analyzed by matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry (MS).…”