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•The roles of amino acid substitutions during the molecular evolution of PAiRFP1 from Agp2 were analyzed.•Residue M163 in PAiRFP1 plays import roles in protein folding.•The residues ...F244 and C280 exerted remarkable effects on ε, Φf, and molecular brightness.•The residues F244 and V276 modulate the maximum absorption and emission peak location.•The reverse mutants V203A, V294E, S218G and D127G possessed better spectral properties than PAiRFP1.
A photoactivatable near-infrared fluorescent protein (NIR-FP) PAiRFP1 has been developed by 15 amino acid substitutions in its nonfluorescent template Agp2. In our previous communication, we investigated the role of three amino acids in PHY domain distal from BV molecule. The impact of the twelve amino acids in GAF domain, especially five residues near BV-binding pocket is unclear. In this paper, PCR based reverse mutagenesis, spectroscopic methods, molecular modelling and simulations have been employed to explore the roles of these substitutions during the molecular evolution of PAiRFP1. It was found that the residue L163 is important for protein folding in PAiRFP1. The residues F244 and C280 exerted remarkable effects on molar extinction coefficient, NIR fluorescence quantum yield, molecular brightness, fluorescence fold, and dark recovery rate. The residues F244 and V276 modulate the maximum absorption and emission peak position. The reverse mutant L168M exhibited a higher fluorescence fold than PAiRFP1. Additionally, the reverse mutants V203A, V294E, S218G and D127G possessed better spectral properties than PAiRFP1. This study is important for the rational design of a better BphP-based photoactivatable NIR-FPs.
Many halogenated compounds have enticed substantial attention as pollutants of environmental concern and pose a serious threat to human health and the environment. To date, halogenated compounds are ...detected by conventional analytical methods, such as chromatography and spectroscopy. Although sensitive and accurate, the traditional methods are highly dependent on expert users, need expensive instrumentation and prolonged analysis time. Hence, rapid, accurate, and in situ detection of these chemicals is crucial to the success of early warning systems. Biosensors for detection of hazardous halocarbons meet many of the requirements for environmental application and have made considerable progress over the last few decades. This review provides a comprehensive overview of biosensors based on dehalogenating enzymes for the detection of halogenated compounds (halocarbon biosensors). The key publications that have shaped the field were included in the study. We discussed the signal transduction principles in halocarbon biosensors and classified them into potentiometric, amperometric, optical, and calorimetric biosensors. We compared halocarbon biosensors in terms of analytical features, bioreceptor (whole-cell, purified enzyme, and mini protein), source organism, immobilization method, and immobilization matrix. We chronologically discussed the progress in halocarbon biosensors and analyzed the development trends. Finally, we highlighted the challenges to halocarbon biosensors and discussed prospects, together with the recommended solutions.
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•Dehalogenases as promising tool for halocarbon detection.•Whole-cells as a biocomponent in halocarbon biosensors remained a common choice.•Optical biosensors have attracted increasing attention.•The projections for the future development of halocarbon biosensors were discussed.
figure: see text The halohydrin dehalogenase from Agrobacterium radiobacter AD1 catalyzed the highly enantioselective and beta-regioselective azidolysis of (substituted) styrene oxides. By means of ...kinetic resolutions the remaining epoxide and the formed azido alcohol could be obtained in very high ee. In a large scale conversion, the decrease in yield and selectivity due to the uncatalyzed chemical side reaction could be overcome by slow addition of azide.
•A method for screening HHDH mutants for the biosynthesis of valuable β-substituted alcohols was developed and demonstrated.•Successful application to a directed evolution experiment demonstrated the ...practical utility of the developed approach.•pH change caused by HHDH-catalyzed epoxide ring-opening was used to develop a high-throughput screening methodology.
Halohydrin dehalogenases (HHDHs) are valuable biocatalysts involved in the synthesis of β-substituted alcohols via their nucleophile-mediated ring-opening activity. To use directed evolution to unleash the latent potential of HHDHs for the synthesis of β-substituted alcohols, we report a high-throughput assay for screening HHDHs mutagenesis libraries. The assay is performed in a 96-well microtiter plate format using a cell-free extract or whole-cells in the presence of the desired nucleophile. The developed method relies upon the color change of bromothymol blue, due to the pH change caused by HHDH-catalyzed ring-opening of the epoxide substrate. The assay was validated using gas chromatography and subsequently applied to high-throughput screening of halohydrin dehalogenase HheC mutagenesis library. Active mutants were found for the tested substrates. Due to its simplicity and flexibility towards the use of nucleophiles and epoxides, the method is an attractive alternative to the existing assays for HHDH epoxide ring-opening reaction and could be helpful in the rapid discovery of industrial biocatalysts.
•The first study indicating Chinese parents’ knowledge on infant crying and AHT.•Nearly half of the parents acknowledged they had ever shaken their babies.•Enriches the literature on the parental ...knowledge about infant crying and AHT.•Enriches the literature by providing the data on shaking behaviors in China.•Underlined the importance of prevention strategies to educate parents about AHT.
This study aims to characterize the knowledge about infant crying and abusive head trauma (AHT), and shaking behaviors in parents of children in China, which are lacking currently.
A cross-sectional survey was conducted in 2020. We collected information about the knowledge of the typical patterns of infant crying and AHT, and asked about beliefs of the effects of violent shaking on children’s health, and shaking behavior among parents.
A total of 568 parents completed the questionnaire, and only 1.6 % of them answered all nine knowledge questions related to infant crying correctly. Overall, 42.6 % of participants reported they had heard about AHT, but only 17.1 % of the parents reported they knew enough about the dangers of infant shaking. About 45 % of the parents acknowledged that they had shaken their infants at least once. Parents who were from western region of China (OR = 3.860; 95 % CI = 1.871, 7.966; p < 0.001) and have felt very frustrated because of the baby's crying over half of the time (OR = 3.401; 95 % CI = 1.862, 6.211; p < 0.001) had the highest risk of shaking.
Majority of the parents reported that they needed further information about infant soothing techniques, knowledge of prevention and treatment about AHT.
Majority of Chinese parents do not have enough knowledge about normal infant crying, nevertheless, most of them expressing needs in learning more.
Community-wide advocating efforts aiming to educate parents on awareness and knowledge about AHT should be a health priority in China.
Halohydrin dehalogenases are bacterial enzymes that catalyse the reversible formation of epoxides from
vicinal halohydrins. A spectrophotometric assay for halohydrin dehalogenases based on the ...absorption difference between the halohydrin
para-nitro-2-bromo-1-phenylethanol and the epoxide
para-nitrostyrene oxide was developed. The enantioselectivity of ring-closure reactions catalysed by three different halohydrin dehalogenases could be estimated from the shape of progress curves. Evaluation of ring-opening reactions catalysed by halohydrin dehalogenase from
Agrobacterium radiobacter AD1 established that, in addition to Cl
− and Br
−, nucleophiles such as N
3
−, CN
− and NO
2
− are also accepted for the ring opening of
para-nitrostyrene oxide. The ring-opening reactions with these nucleophiles resulted in highly enantioselective kinetic resolutions, which expands the scope of synthetically valuable conversions catalysed by a halohydrin dehalogenase.
Graphic
As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin ...dehalogenase from
Agrobacterium radiobacter
AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (
t
1/2
) inactivation at 65 °C, 18 °C increase in apparent
T
m
value, and 20 °C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of
rac
-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.
Biocatalytic transformations in organic synthesis often require the use of organic solvents to improve substrate solubility and promote the product formation. Halohydrin dehalogenases (HHDHs) are ...enzymes that catalyze the formation and conversion of epoxides, important synthetic class of compounds that are often sparingly soluble in water and prone to hydrolysis. In this study, the activity, stability, and enantioselectivity of HHDH from
Agrobacterium radiobacter
AD1 (HheC) in form of cell-free extract were evaluated in various aqueous-organic media. A correlation was discovered between the enzyme activity in the ring-closure reaction and logP of the solvent. Knowledge of such a relationship makes biocatalysis with organic solvents more predictable, which may reduce the need to experiment with a variety of solvents in the future. The results revealed a high enzyme compatibility with hydrophobic solvents (e.g.,
n
-heptane) in terms of activity and stability. Regarding the HHDH applicability in an organic medium, inhibitions by a number of solvents (e.g., THF, toluene, chloroform) proved to be a more challenging problem than the protein stability, especially in the ring-opening reaction, thus suggesting which solvents should be avoided. In addition, solvent tolerance of the thermostable variant ISM-4 was also evaluated, revealing increased stability and to a lesser extent enantioselectivity compared to the wild-type. This is the first time such a systematic analysis has been reported, giving insight into the behavior of HHDHs in nonconventional media and opening new opportunities for the future biocatalytic applications.
Key points
•
HheC performs better in the presence of hydrophobic than hydrophilic solvents.
•
Enzyme activity in the PNSHH ring-closure reaction is a function of the logP.
•
Thermostability of ISM-4 variant is accompanied by superior solvent tolerance.
Graphical Abstract
Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 is a homotetrameric protein containing four tryptophan residues per subunit. The fluorescence properties of the enzyme are strongly ...influenced by halide binding. To examine the role of the tryptophans (W139, W192, W238, and W249) in halide binding and catalysis, they were individually mutated to a phenylalanine. All mutations, except for W238F, influenced the enzymatic properties. Mutating W192 to phenylalanine inactivated the enzyme and led to dissociation into dimers and monomers. In the structure of HheC, residue W139 and residue W249 from the opposite subunit are close to the active site of the enzyme. Substitution of W139 mainly affected K(m) values with all tested substrates and reduced the enantiopreference for p-nitro-2-bromo-1-phenylethanol. Replacing W249 increased both k(cat) and K(m) values with all tested substrates except for the (S)-enantiomer of p-nitro-2-bromo-1-phenylethanol, for which k(cat) was 3-fold decreased, resulting in a 6-fold increase of the enantioselectivity. Fluorescence measurements revealed that in the ligand-free state the intrinsic protein fluorescence of mutant W139F is higher than that of the wild-type enzyme, while the fluorescence intensity of mutants W238F and W249F was lower. The fluorescence intensities of the W238F and W249F enzymes were increased when they were unfolded or when bromide was added, whereas the fluorescence of mutant W139F was not increased by unfolding or addition of bromide. These results demonstrate that the fluorescence of residues W238 and W249 is partially quenched in the folded ligand-free state, and that W139 is completely quenched and acts as an energy acceptor for the other tryptophan residues as well. Changes of the maximum fluorescence emission wavelength of the HheC variants and the results of acrylamide quenching experiments confirmed that bromide binding induces a local conformational change around the active site, resulting in residue W139 and the quencher group being separated.
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