Biology subjects

Vinogradov, A. A.

Publications and source records attributed to Vinogradov, A. A..

5 recordsLinked to original sources

bioRxiv2025

Aminoacyl-tRNA specificity of a ligase catalyzing non-ribosomal peptide extension

Peptide aminoacyl-transfer ribonucleic acid ligases (PEARLs) are amide bond-forming enzymes that extend the main chain of peptides using aminoacyl-tRNA (aa-tRNA) as a substrate. In this study, we investigated the substrate specificity of the PEARL BhaBCAla from Bacillus halodurans, which utilizes Ala-tRNAAla. By leveraging flexizyme, a ribozyme capable of charging diverse acids onto a desired tRNA, we generated an array of aa-tRNAs in which we varied both the amino acid and the tRNA to dissect the substrate scope of BhaBCAla. We demonstrate that BhaBCAla catalyzes peptide extension with non-cognate proteinogenic and non-canonical amino acids, hydroxy acids, and mercaptocarboxylic acids when attached to tRNAAla. For most of these, the efficiency was considerably reduced compared to Ala, indicating the enzyme recognizes the amino acid. By varying the different parts of the tRNA, enzyme specificity was shown to also depend on the acceptor stem and the anticodon arm of the tRNA. These findings establish the molecular determinants of PEARL specificity and provide a foundation for engineering these enzymes for broader applications in peptide synthesis.

biochemistry↗

bioRxiv2024

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of anti-malarial therapeutics.

biophysics↗

bioRxiv2022

Accurate models of substrate preferences of post-translational modification enzymes from a combination of mRNA display and deep learning

Promiscuous post-translational modification (PTM) enzymes often display non-obvious substrate preferences by acting on diverse yet well-defined sets of peptides and/or proteins. Thorough understanding of substrate fitness landscapes for promiscuous PTM enzymes is important because they play key roles in many areas of contemporary science, including natural product biosynthesis, molecular biology and biotechnology. Here, we report the development of an integrated platform for accurate profiling of substrate preferences for PTM enzymes. The platform features a combination of i) mRNA display with next generation sequencing as an ultrahigh throughput technique for data acquisition and ii) deep learning for data analysis. The high accuracy (>0.99 in each of two studies) and generalizability of the resulting deep learning models enables comprehensive analysis of enzymatic substrate preferences. The models can be utilized to quantify fitness across sequence space, map modification sites, and identify important amino acids in the substrate. To benchmark the platform, we perform substrate specificity profiling of a Ser dehydratase (LazBF) and a Cys/Ser cyclodehydratase (LazDEF), two enzymes from the lactazole biosynthesis pathway. In both studies, our results point to highly complex enzymatic preferences, which, particularly for LazBF, cannot be reduced to a set of simple rules. The ability of the constructed models to dissect and analyze such complexity suggests that the developed platform can facilitate the wider study of PTM enzymes.

biochemistry↗

bioRxiv2020

Promiscuous enzymes cooperate at the substrate level en route to lactazole A

Enzymes involved in ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis often have relaxed specificity profiles and are able to modify diverse substrates. When several such enzymes act together during precursor peptide maturation, a multitude of products can form, and yet usually, the biosynthesis converges on a single natural product. For the most part, the mechanisms controlling the integrity of RiPP assembly remain elusive. Here, we investigate biosynthesis of lactazole A, a model thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of biosynthetic events at the individual modification level, and supplement this study with substrate scope analysis for participating enzymes. Combined, our results reveal a dynamic thiopeptide assembly process with multiple points of kinetic control, intertwined enzymatic action, and the overall substrate-level cooperation between the enzymes. This work advances our understanding of RiPP biosynthesis processes and facilitates thiopeptide bioengineering.

biochemistry↗

bioRxiv2019

Minimal lactazole scaffold for in vitro production of pseudo-natural thiopeptides

Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, we established a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis revealed remarkable substrate tolerance of the biosynthetic enzymes, and led to the development of the \"minimal lactazole scaffold\", a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz enabled access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and up to 18 amino acid-long tail regions. Moreover, utilizing genetic code reprogramming, we demonstrated synthesis of pseudo-natural lactazoles containing 4 non-proteinogenic amino acids. This work opens possibilities in exploring novel sequence space of pseudo-natural thiopeptides.

biochemistry↗