Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor

Zhang, Yan; Pan, Jiang; Luan, Zheng-Jiao; Xu, Guochao; Park, Sunghoon; Xu, Jian‐He

doi:10.1128/aem.01597-14

Cited by 18 publications

(22 citation statements)

References 39 publications

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“…In our previous work, a carboxylic esterase RhEst1 was identified and isolated from Rhodococcus sp. ECU1013, which can catalyze the hydrolysis of S-DmCpCe with high enantioselectivity [12,13] (Fig. S1).…”

Section: Several Efforts Have Been Reported Towards Biocatalytic Syntmentioning

confidence: 99%

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Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

Chen

Luan

et al. 2015

Journal of Molecular Graphics and Modelling

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A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. This work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications.

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Section: Several Efforts Have Been Reported Towards Biocatalytic Syntmentioning

confidence: 99%

“…Cultivation of the recombinant E. coli cells expressing RhEst1 or its mutants and enzyme purification using Ni 2+ affinity chromatography were performed as described previously [13,32,33].…”

Section: Site-directed Mutations Were Constructed By Quick Change® Simentioning

confidence: 99%

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

Chen

Luan

et al. 2015

Journal of Molecular Graphics and Modelling

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“…Detoxifying proteins, such as penicillin binding proteins, are also presumed to be located mostly in the periplasm. [60,61] are, however, present in this strain.…”

Section: Protein Location In the Cellmentioning

confidence: 55%

The complete genome sequence of the nitrile biocatalyst Rhodocccus rhodochrous ATCC BAA-870

Frederick

Hennessy

Horn

et al. 2019

Preprint

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Background Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst. Results The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci. Conclusions The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.

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“…sp. ECU1013 (RhEst1) was shown to hydrolyze rac -ethyl-2,2-dimethyl- c -propanecarboxylate ( 94 , rac-DMCPCM) to give ( S )-(+)-2,2-dimethyl-c-propylcarboxylic acid ( 96 , ( S )-DMCPCA)—a valuable precursor in the synthesis of the drug cilastatin ( 97 ) (Scheme 24) [214,215]. This shows an alternative route to cilastatin compared to the previously mentioned nitrile hydratase-catalyzed process starting with 2,2-dimethyl-c-propanecarbonitrile [136].…”

Section: Hydrolase Activity In Rhodococcusmentioning

confidence: 99%

“…Enantioselective resolution of a racemic mixture of DMCPCM ( 94 ) to yield ( S )-DMCPCA ( 96 )—a precursor for the drug cilastatin ( 97 ) [214]. …”

Section: Figure and Schemesmentioning

confidence: 99%

Rhodococcus as a Versatile Biocatalyst in Organic Synthesis

Busch

Hagedoorn

Hanefeld

2019

IJMS

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The application of purified enzymes as well as whole-cell biocatalysts in synthetic organic chemistry is becoming more and more popular, and both academia and industry are keen on finding and developing novel enzymes capable of performing otherwise impossible or challenging reactions. The diverse genus Rhodococcus offers a multitude of promising enzymes, which therefore makes it one of the key bacterial hosts in many areas of research. This review focused on the broad utilization potential of the genus Rhodococcus in organic chemistry, thereby particularly highlighting the specific enzyme classes exploited and the reactions they catalyze. Additionally, close attention was paid to the substrate scope that each enzyme class covers. Overall, a comprehensive overview of the applicability of the genus Rhodococcus is provided, which puts this versatile microorganism in the spotlight of further research.

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Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor

Cited by 18 publications

References 39 publications

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

The complete genome sequence of the nitrile biocatalyst Rhodocccus rhodochrous ATCC BAA-870

Rhodococcus as a Versatile Biocatalyst in Organic Synthesis

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