scholarly journals Bacterial cytochrome P450-catalyzed regio- and stereoselective steroid hydroxylation enabled by directed evolution and rational design

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Xiaodong Zhang ◽  
Yaqin Peng ◽  
Jing Zhao ◽  
Qian Li ◽  
Xiaojuan Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Cytochrome P450 ◽  
Directed Evolution ◽  
Rational Design ◽  
Cytochrome P450s ◽  
Steroid Hydroxylation ◽  
Recent Developments ◽  
Low Activity ◽  
High Expression Level

AbstractSteroids are the most widely marketed products by the pharmaceutical industry after antibiotics. Steroid hydroxylation is one of the most important functionalizations because their derivatives enable a higher biological activity compared to their less polar non-hydroxylated analogs. Bacterial cytochrome P450s constitute promising biocatalysts for steroid hydroxylation due to their high expression level in common workhorses like Escherichia coli. However, they often suffer from wrong or insufficient regio- and/or stereoselectivity, low activity, narrow substrate range as well as insufficient thermostability, which hampers their industrial application. Fortunately, these problems can be generally solved by protein engineering based on directed evolution and rational design. In this work, an overview of recent developments on the engineering of bacterial cytochrome P450s for steroid hydroxylation is presented.

Function and engineering of the 15β-hydroxylase CYP106A2

2006 ◽  
Vol 34 (6) ◽  
pp. 1215-1218 ◽  
Author(s):  
C. Virus ◽  
M. Lisurek ◽  
B. Simgen ◽  
F. Hannemann ◽  
R. Bernhardt
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Steroid Hormones ◽  
Bacillus Megaterium ◽  
Crystallization Process ◽  
High Yield ◽  
Recent Developments ◽  
Adrenodoxin Reductase ◽  
Hydroxylation Activity

CYP106A2 from Bacillus megaterium ATCC 13368 is a bacterial cytochrome P450 that is capable of transforming steroid hormones. It can be easily expressed in Escherichia coli with a high yield. Its activity in vitro can be achieved by using the adrenal redox proteins adrenodoxin and adrenodoxin reductase. So far, it was not possible to crystallize CYP106A2 because of degradation during the crystallization process. Nevertheless, CYP106A2 is an interesting enzyme for biotechnological use. It hydroxylates pharmaceutically important steroids such as progesterone and 11-deoxycortisol. However, it will be necessary for efficient application of CYP106A2 in biotechnology to improve the hydroxylation activity and manipulate the regiospecificity. The present paper gives an overview of recent developments in protein engineering of CYP106A2.

scholarly journals Engineering the biomimetic cofactors of NMNH for cytochrome P450 BM3 based on binding conformation refinement

RSC Advances ◽  
2021 ◽  
Vol 11 (20) ◽  
pp. 12036-12042
Author(s):  
Yao Liu ◽  
Yalong Cong ◽  
Chuanxi Zhang ◽  
Bohuan Fang ◽  
Yue Pan ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Rational Design ◽  
Design Strategy ◽  
Efficient Utilization ◽  
P450 Bm3 ◽  
Binding Conformation ◽  
Cytochrome P450 Bm3

A rational design strategy was proposed to improve the efficient utilization of alternative biomimetic cofactor by P450 BM3 enzyme.

scholarly journals Recent developments of synthesis and biological activity of sultone scaffolds in medicinal chemistry

2021 ◽  
Vol 14 (4) ◽  
pp. 103037
Author(s):  
Yingying Xu ◽  
Ziwen Zhang ◽  
Jingbo Shi ◽  
Xinhua Liu ◽  
Wenjian Tang
Keyword(s):  
Biological Activity ◽  
Medicinal Chemistry ◽  
Recent Developments

scholarly journals Photochemical Reactivity of Naphthol-Naphthalimide Conjugates and Their Biological Activity

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3355
Author(s):  
Matija Sambol ◽  
Patricia Benčić ◽  
Antonija Erben ◽  
Marija Matković ◽  
Branka Mihaljević ◽  
...  
Keyword(s):  
Biological Activity ◽  
Rational Design ◽  
Human Cancer ◽  
Photophysical Properties ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Quinone Methide ◽  
Quantum Yields ◽  
Photochemical Reactivity ◽  
Human Cancer Cell Lines

Quinone methide precursors 1a–e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (ΦF ≈ 10−2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (ΦR ≈ 10−5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants Ka ≈ 105–106 M−1, as well as with bovine serum albumin (BSA) Ka ≈ 105 M−1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a–e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

Cytokinin activity of some substituted ureas and thioureas

1966 ◽  
Vol 165 (999) ◽  
pp. 245-265 ◽  
Keyword(s):  
Biological Activity ◽  
Active Compound ◽  
Phenyl Ring ◽  
Chemical Structure ◽  
Lower Activity ◽  
Cytokinin Activity ◽  
Hydrogen Atoms ◽  
Detectable Activity ◽  
Low Activity

N , N '-Diphenylurea was shown to have reproducible cytokinin activity . Some 500 ureas, mainly of the N -monosubstituted and N , N '-disubstituted types, were tested an d about one half of these were active. Attempts were made to correlate chemical structure with biological activity. Although there are some exceptions to nearly every generalization it has been possible to formulate some principles. (1) Phenyl urea was the simplest active compound. (2) An HNCONH bridge conferred higher activity than an HNCSNH linkage and any other tested arrangement of the bridge gave inactive com pounds. (3) Compounds in which both amino hydrogen atoms on one or both sides of the bridge were substituted were of low activity or were inactive. (4) Many com pounds of the type R NHCONH 2 in which R = a substituted phenyl ring were tested. Ring substitution generally increased the activity and the highest activity was associated with meta substitution and the lowest with ortho . Compounds with electron-attracting substituents were generally more active than those with electron-donating substituents. Pyridyl compounds were active but com pounds with non-planar rings were inactive. (5) In compounds of the type R NHCONH R ' in which R and R ' were phenyl or substituted phenyl groups the highest activities were usually found in com pounds with one unsubstituted phenyl ring. Those with two substituted phenyl groups generally had lower activity. Some ureas showed detectable activity at 0.1 parts/10 6 . This was about four times less active than kinetin when tested in the tobacco pith assay.

scholarly journals Recent developments in the synthesis and biological activity of acridine/acridone analogues

RSC Advances ◽  
2017 ◽  
Vol 7 (26) ◽  
pp. 15776-15804 ◽  
Author(s):  
Monika Gensicka-Kowalewska ◽  
Grzegorz Cholewiński ◽  
Krystyna Dzierzbicka
Keyword(s):  
Biological Activity ◽  
The World ◽  
Recent Developments

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