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Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
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Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
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Updated: Feb 28, 2026

Defining Substrate Specificities for Lipase and Phospholipase Candidates
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Decodificación del panorama de especificidad de sustrato de una enzima promiscua mediante escaneo mutacional

Rosario Vanella1,2, Sean Boult3,4,5, Christoph Küng3,4

  • 1Department of Chemistry, University of Basel, Basel, Switzerland. rosario.vanella@unibas.ch.

Nature communications
|February 26, 2026
PubMed
Resumen

La secuenciación de proximidad de enzimas (EP-Seq) decodifica cómo las mutaciones alteran la especificidad del sustrato de la oxidasa de D-aminoácidos (DAOx). Esto revela estrategias para diseñar enzimas altamente selectivas.

Palabras clave:
EnzimologíaIngeniería de proteínasBiocatálisisEspecificidad de sustratoOxidasa de D-aminoácidosMutagénesisSecuenciación de proximidad de enzimasDiseño de enzimasInteligencia artificial

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Área de la Ciencia:

  • Enzimología
  • Ingeniería de proteínas
  • Biocatálisis

Sus antecedentes:

  • La especificidad del sustrato de la enzima es crucial pero difícil de entender e ingeniar a nivel molecular.
  • La oxidasa de D-aminoácidos (DAOx) de Rhodotorula gracilis sirve como enzima modelo promiscua para estudiar la especificidad.

Objetivo del estudio:

  • Mapear sistemáticamente cómo las mutaciones afectan las preferencias de sustrato de DAOx.
  • Identificar los mecanismos subyacentes a la determinación de la especificidad y guiar la ingeniería de enzimas.

Principales métodos:

  • Se utilizó la secuenciación de proximidad de enzimas (EP-Seq) para generar ~40,000 pares de secuencias-fenotipos.
  • Se evaluaron ~6,500 variantes de DAOx frente a cinco sustratos de D-aminoácidos con propiedades variables.

Principales resultados:

  • Se identificaron mutaciones específicas del sustrato distribuidas por toda la estructura de la enzima.
  • Las mutaciones del sitio activo alteran drásticamente la especificidad pero reducen la actividad; las mutaciones distales modulan sutilmente la especificidad con una pérdida mínima de actividad.
  • Se descubrieron puntos calientes alostéricos que influyen en la especificidad y se caracterizaron variantes con especificidad exclusiva o grandes cambios de preferencia (hasta 230 veces).

Conclusiones:

  • Se estableció un marco sólido para decodificar la especificidad de las enzimas utilizando la caracterización de variantes a gran escala.
  • Se demostró que la combinación de mutaciones complementarias mejora la discriminación del sustrato para el diseño racional de biocatalizadores.
  • Se proporcionaron conjuntos de datos fundamentales para avanzar en la ingeniería de enzimas guiada por IA.