Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase

2021 ◽  
Author(s):  
Feng Cheng ◽  
Qiu‐Yao Zhai ◽  
Xiao‐Fan Gao ◽  
Hua‐Tao Liu ◽  
Shuai Qiu ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Carbonyl Reductase ◽  
Enzymatic Properties

scholarly journals Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Min Li ◽  
Zhi-Jun Zhang ◽  
Xu-Dong Kong ◽  
Hui-Lei Yu ◽  
Jiahai Zhou ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Engineering ◽  
Directed Evolution ◽  
Specific Activity ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Carbonyl Reductase ◽  
Triple Mutant ◽  
Engineering Strategy ◽  
Substrate Tolerance

ABSTRACT Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T 50 15, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C 50 15, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter−1 day−1. These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor. IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

Enzymatic properties of mouse 25-hydroxyvitamin D3 1α-hydroxylase expressed in Escherichia coli

2001 ◽  
Vol 259 (3) ◽  
pp. 731-738 ◽  
Author(s):  
Toshiyuki Sakaki ◽  
Natsumi Sawada ◽  
Ken-ichi Takeyama ◽  
Shigeaki Kato ◽  
Kuniyo Inouye
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Properties

Protein Engineering of Penicillin Acylase

Acta Naturae ◽  
2010 ◽  
Vol 2 (3) ◽  
pp. 47-61 ◽  
Author(s):  
V I Tishkov ◽  
S S Savin ◽  
A S Yasnaya
Keyword(s):  
Protein Engineering ◽  
Penicillin Acylase

Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei.

1977 ◽  
Vol 24 (2) ◽  
pp. 423-435 ◽  
Author(s):  
O Brison ◽  
C Kedinger ◽  
J Wilhelm
Keyword(s):  
Viral Replication ◽  
Hela Cell ◽  
Adenovirus Type ◽  
Enzymatic Properties ◽  
Cell Nuclei ◽  
Adenovirus Type 2

scholarly journals Flavin Adenine Dinucleotide-Dependent Halogenase XanH and Engineering of Multifunctional Fusion Halogenases

2020 ◽  
Vol 86 (18) ◽  
Author(s):  
Lingxin Kong ◽  
Qing Wang ◽  
Zixin Deng ◽  
Delin You
Keyword(s):  
Protein Engineering ◽  
Fusion Protein ◽  
Natural Product ◽  
Flavin Adenine Dinucleotide ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Good Alternative ◽  
Limited Information ◽  
Flavin Reductase ◽  
Free Standing

ABSTRACT Xantholipin (compound 1), a polycyclic xanthone antibiotic, exhibited strong antibacterial activities and showed potent cytotoxicity. The biosynthetic gene cluster of compound 1 has been identified in our previous work, and the construction of xanthone nucleus has been well demonstrated. However, limited information of the halogenation involved in compound 1 biosynthesis is available. In this study, based on the genetic manipulation and biochemical assay, we characterized XanH as an indispensable flavin adenine dinucleotide (FAD)-dependent halogenase (FDH) for the biosynthesis of compound 1. XanH was found to be a bifunctional protein capable of flavin reduction and chlorination and exclusively used the NADH. However, the reduced flavin could not be fully and effectively utilized, and the presence of an extra flavin reductase (FDR) and chemical-reducing agent could promote the halogenation. XanH accepted its natural free-standing substrate with angular fused polycyclic aromatic systems. Meanwhile, it exhibited moderate halogenation activity and possessed high substrate specificity. The requirement of extra FDR for higher halogenation activity is tedious for future engineering. To facilitate efforts in engineering XanH derivative proteins, we constructed the self-sufficient FDR-XanH fusion proteins. The fusion protein E1 with comparable activities to that of XanH could be used as a good alternative for future protein engineering. Taken together, these findings reported here not only improve the understanding of polycyclic xanthones biosynthesis but also expand the substrate scope of FDH and pave the way for future engineering of biocatalysts for new active substance synthesis. IMPORTANCE Halogenation is important in medicinal chemistry and plays an essential role in the biosynthesis of active secondary metabolites. Halogenases have evolved to catalyze reactions with high efficiency and selectivity, and engineering efforts have been made to engage the selective reactivity in natural product biosynthesis. The enzymatic halogenations are an environmentally friendly approach with high regio- and stereoselectivity, which make it a potential complement to organic synthesis. FDHs constitute one of the most extensively elucidated class of halogenases; however, the inventory awaits to be expanded for biotechnology applications and for the generation of halogenated natural product analogues. In this study, XanH was found to reduce flavin and halogenated the freely diffusing natural substrate with an angular fused hexacyclic scaffold, findings which were different from those for the exclusively studied FDHs. Moreover, the FDR-XanH fusion protein E1 with comparable reactivity to that of XanH serves as a successful example of genetic fusions and sets an important stage for future protein engineering.

scholarly journals Adult human liver contains intermediate-type proteasomes with different enzymatic properties

2014 ◽  
Vol 13 (4) ◽  
pp. 429-438 ◽  
Author(s):  
Sabrina Gohlke ◽  
Alexander Kloβ ◽  
Michael Tsokos ◽  
Kathrin Textoris-Taube ◽  
Christin Keller ◽  
...  
Keyword(s):  
Human Liver ◽  
Enzymatic Properties ◽  
Intermediate Type ◽  
Adult Human
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