Improved X‐ray diffraction from Bacillus megaterium penicillin G acylase crystals through long cryosoaking dehydration
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TLDR
In this paper, a crystal of Bacillus megaterium (BmPGA) was diffracted X-rays to 2.20"A resolution and belonged to the monoclinic space group P21 with one molecule of BmPGAs in the asymmetric unit.Abstract:
Penicillin G acylase from Bacillus megaterium (BmPGA) is currently used in the pharmaceutical industry as an alternative to PGA from Escherichia coli (EcPGA) for the hydrolysis of penicillin G to produce 6-aminopenicillanic acid (6-APA), a penam nucleus for semisynthetic penicillins. Despite the significant differences in amino-acid sequence between PGAs from Gram-positive and Gram-negative bacteria, a representative PGA structure of Gram-positive origin has never been reported. In this study, crystallization and diffraction studies of BmPGA are described. Poor diffraction patterns with blurred spots at higher resolution were typical for BmPGA crystals cryocooled after a brief immersion in cryoprotectant solution. Overnight soaking in the same cryo-solution substantially improved both the mosaicity and resolution limit through the establishment of a new crystal-packing equilibrium. A crystal of BmPGA diffracted X-rays to 2.20 A resolution and belonged to the monoclinic space group P21 with one molecule of BmPGA in the asymmetric unit.read more
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Increasing the X-ray diffraction power of protein crystals by dehydration: the case of bovine serum albumin and a survey of literature data.
TL;DR: Dehydration was found to increase the diffraction limit of BSA crystals from ~8 Å to 3.2 Å, probably by improving the packing of protein molecules in the crystal lattice, and it can be a useful procedure to be used in initial screening as a method of improving the diffractions limits of existing crystals.
Journal ArticleDOI
Visualized characterization of bacterial penicillin G acylase for the hydrolysis of β-lactams using an activatable NIR fluorescent probe
Zhenhao Tian,Zhenhao Tian,Lei Feng,Lei Feng,Lu Li,Xiangge Tian,Xiangge Tian,Jingnan Cui,Hou-Li Zhang,Chao Wang,Hui-Lian Huang,Baojing Zhang,Xiaochi Ma +12 more
TL;DR: An activated off-on near-infrared (NIR) fluorescent probe (DDAP) deduced from a novel fluorophore has been designed and developed for sensitively and selectively detecting endogenous PGA in real-time and could be applied to identify bacteria with PGA expression from complex microorganism samples.
ComponentDOI
Crystal structures and protein engineering of three different penicillin G acylases from Gram-positive bacteria with different thermostability.
Janine Mayer,Jan Pippel,Gabriele Günther,Carolin Müller,Anna Lauermann,Tobias Knuuti,Wulf Blankenfeldt,Dieter Jahn,Rebekka Biedendieck +8 more
TL;DR: The first three crystal structures for PGAs from Gram-positive Bacilli and their utilization in protein engineering experiments for the manipulation of their thermostability provide the basis for establishment of new PGA-based biotechnological processes.
Journal ArticleDOI
An evaluation of adhesive sample holders for advanced crystallographic experiments.
TL;DR: Adhesives use as tools for advanced sample mounting and cryoprotection is assessed and their suitability for room-temperature data-collection and humidity-controlled studies is investigated.
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