134003-04-6Relevant articles and documents
Efficient synthesis of the intermediate of abacavir and carbovir using a novel (+)-γ-lactamase as a catalyst
Gao, Shuaihua,Zhu, Shaozhou,Huang, Rong,Lu, Yingxiu,Zheng, Guojun
, p. 3878 - 3881 (2015)
Abstract The enantiomers of 2-azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) are key chiral synthons in the synthesis of antiviral drugs such as carbovir and abacavir. (+)-γ-Lactamase can be used as a catalyst in the enzymatic preparation of optically pure (-)-γ-lactam. Here, a (+)-γ-lactamase discovered from Bradyrhizobium japonicum USDA 6 by sequence-structure guided genome mining was cloned, purified and characterized. The enzyme possesses a significant catalytic activity towards γ-lactam. The active site of the (+)-γ-lactamase was studied by homologous modeling and molecular docking, and the accuracy of the prediction was confirmed by site-specific mutagenesis. The (+)-γ-lactamase reveals the great practical potential as an enzymatic method for the efficient production of carbocyclic nucleosides of pharmaceutical interest.
Novel screening methods - The key to cloning commercially successful biocatalysts
Taylor, Stephen J. C.,Brown, Rob C.,Keene, Phil A.,Taylor, Ian N.
, p. 2163 - 2168 (1999)
Providing sufficient biocatalyst to support the demands of multi tonne product supply can be problematical. Here we describe how screening for and cloning a γ-lactamase overcame biocatalyst supply issues, and greatly improved the actual biocatalytic process. The isolation of an expressing γ- lactamase clone from a gene library necessitated a combination of classical molecular biology techniques together with innovative screening methods to identify a functional clone. Once isolated the enzyme was characterised with regard to its process performance and proved to be active at 500 g L-1 substrate. Further development of the recombinant fermentation and downstream processing has resulted in the ability to produce sufficient biocatalyst from one 500 l fermentation to resolve 5 metric tonnes of (±)-lactam, whilst simplifying the process chemistry greatly.
Stereo- and regiocontrolled synthesis of highly functionalized cyclopentanes with multiple chiral centers
Nonn, Melinda,Binder, Adrienn,Volk, Balázs,Kiss, Loránd
, p. 1199 - 1209 (2020/03/17)
The synthesis of some highly substituted three-dimensional cyclopentanes with multiple chiral centers and with high regiochemical and stereochemical diversity has been accomplished starting from cyclopentadiene-derived aminocyclopentenecarboxylic acids. The small-molecular design consisted of stereo- and regiocontrolled functionalization of the starting cyclopentene β- and γ-amino acids through oxirane formation/oxirane opening and afforded regio- and diastereoisomers of orthogonally protected aminocyclopentanecarboxylates.
Catalytic Promiscuity of Ancestral Esterases and Hydroxynitrile Lyases
Devamani, Titu,Rauwerdink, Alissa M.,Lunzer, Mark,Jones, Bryan J.,Mooney, Joanna L.,Tan, Maxilmilien Alaric O.,Zhang, Zhi-Jun,Xu, Jian-He,Dean, Antony M.,Kazlauskas, Romas J.
supporting information, p. 1046 - 1056 (2016/02/05)
Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ～100 million years ago. Both enzyme types are α/β-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.
