Modelling of substrate access and substrate binding to cephalosporin acylases

Abstract Semisynthetic cephalosporins are widely used antibiotics currently produced by different chemical steps under harsh conditions, which results in a considerable amount of toxic waste. Biocatalytic synthesis by the cephalosporin acylase from Pseudomonas sp. strain N176 is a promising alternat...

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Autores principales:	Valerio Ferrario, Mona Fischer, Yushan Zhu, Jürgen Pleiss
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2019
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6ec7e748467a4151899601d6033f6264
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spelling	oai:doaj.org-article:6ec7e748467a4151899601d6033f62642021-12-02T15:09:54ZModelling of substrate access and substrate binding to cephalosporin acylases10.1038/s41598-019-48849-z2045-2322https://doaj.org/article/6ec7e748467a4151899601d6033f62642019-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-019-48849-zhttps://doaj.org/toc/2045-2322Abstract Semisynthetic cephalosporins are widely used antibiotics currently produced by different chemical steps under harsh conditions, which results in a considerable amount of toxic waste. Biocatalytic synthesis by the cephalosporin acylase from Pseudomonas sp. strain N176 is a promising alternative. Despite intensive engineering of the enzyme, the catalytic activity is still too low for a commercially viable process. To identify the bottlenecks which limit the success of protein engineering efforts, a series of MD simulations was performed to study for two acylase variants (WT, M6) the access of the substrate cephalosporin C from the bulk to the active site and the stability of the enzyme-substrate complex. In both variants, cephalosporin C was binding to a non-productive substrate binding site (E86α, S369β, S460β) at the entrance to the binding pocket, preventing substrate access. A second non-productive binding site (G372β, W376β, L457β) was identified within the binding pocket, which competes with the active site for substrate binding. Noteworthy, substrate binding to the protein surface followed a Langmuir model resulting in binding constants K = 7.4 and 9.2 mM for WT and M6, respectively, which were similar to the experimentally determined Michaelis constants KM = 11.0 and 8.1 mM, respectively.Valerio FerrarioMona FischerYushan ZhuJürgen PleissNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 9, Iss 1, Pp 1-13 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Valerio Ferrario Mona Fischer Yushan Zhu Jürgen Pleiss Modelling of substrate access and substrate binding to cephalosporin acylases
description	Abstract Semisynthetic cephalosporins are widely used antibiotics currently produced by different chemical steps under harsh conditions, which results in a considerable amount of toxic waste. Biocatalytic synthesis by the cephalosporin acylase from Pseudomonas sp. strain N176 is a promising alternative. Despite intensive engineering of the enzyme, the catalytic activity is still too low for a commercially viable process. To identify the bottlenecks which limit the success of protein engineering efforts, a series of MD simulations was performed to study for two acylase variants (WT, M6) the access of the substrate cephalosporin C from the bulk to the active site and the stability of the enzyme-substrate complex. In both variants, cephalosporin C was binding to a non-productive substrate binding site (E86α, S369β, S460β) at the entrance to the binding pocket, preventing substrate access. A second non-productive binding site (G372β, W376β, L457β) was identified within the binding pocket, which competes with the active site for substrate binding. Noteworthy, substrate binding to the protein surface followed a Langmuir model resulting in binding constants K = 7.4 and 9.2 mM for WT and M6, respectively, which were similar to the experimentally determined Michaelis constants KM = 11.0 and 8.1 mM, respectively.
format	article
author	Valerio Ferrario Mona Fischer Yushan Zhu Jürgen Pleiss
author_facet	Valerio Ferrario Mona Fischer Yushan Zhu Jürgen Pleiss
author_sort	Valerio Ferrario
title	Modelling of substrate access and substrate binding to cephalosporin acylases
title_short	Modelling of substrate access and substrate binding to cephalosporin acylases
title_full	Modelling of substrate access and substrate binding to cephalosporin acylases
title_fullStr	Modelling of substrate access and substrate binding to cephalosporin acylases
title_full_unstemmed	Modelling of substrate access and substrate binding to cephalosporin acylases
title_sort	modelling of substrate access and substrate binding to cephalosporin acylases
publisher	Nature Portfolio
publishDate	2019
url	https://doaj.org/article/6ec7e748467a4151899601d6033f6264
work_keys_str_mv	AT valerioferrario modellingofsubstrateaccessandsubstratebindingtocephalosporinacylases AT monafischer modellingofsubstrateaccessandsubstratebindingtocephalosporinacylases AT yushanzhu modellingofsubstrateaccessandsubstratebindingtocephalosporinacylases AT jurgenpleiss modellingofsubstrateaccessandsubstratebindingtocephalosporinacylases
_version_	1718387758403158016

Modelling of substrate access and substrate binding to cephalosporin acylases

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