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Amines to Amides: Acylation of Amines01:19

Amines to Amides: Acylation of Amines

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Various carboxylic acid derivatives (such as acid chlorides, esters, and anhydrides) can be used for the acylation of amines to yield amides. The reaction requires two equivalents of amines. The first amine molecule functions as a nucleophile and attacks the carbonyl carbon to produce a tetrahedral intermediate. This is followed by the loss of the leaving group and restoration of the C=O bond.
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Enamine formation involves the addition of carbonyl compounds to a secondary amine through a series of reactions. The mechanism begins with the generation of carbinolamine, a nucleophilic attack followed by several proton transfer reactions. The hydroxyl group of the carbinolamine is converted into water to make a better leaving group that can push the reaction forward by eliminating a water molecule. In enamine formation, the last step involves the abstraction of a proton from the α carbon to...
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Indirect-acting cholinergic agonists work by interacting with an enzyme called acetylcholinesterase (AChE) in the synaptic cleft. They can be reversible or irreversible inhibitors and have different effects on the enzyme.
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Reduction of Alkenes: Asymmetric Catalytic Hydrogenation02:17

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Catalytic hydrogenation of alkenes is a transition-metal catalyzed reduction of the double bond using molecular hydrogen to give alkanes. The mode of hydrogen addition follows syn stereochemistry.
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Structure of Amines01:19

Structure of Amines

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The hybridized nitrogen atom in amines possesses a lone pair of electrons and is bound to three substituents with a bond angle of around 108°, which is less than the tetrahedral angle of 109.5°. However, the C–N–H bond angle is slightly larger at 112°, with a carbon–nitrogen bond length of 147 pm. This carbon–nitrogen bond length of of amines is longer than the carbon–oxygen bond of alcohols (143 pm) but shorter than alkanes’...
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Amides to Amines: LiAlH4 Reduction01:20

Amides to Amines: LiAlH4 Reduction

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Amide reduction with strong reducing agents like lithium aluminum hydride proceeds through a nucleophilic acyl substitution to form amines. Primary, secondary, and tertiary amides yield primary, secondary, and tertiary amines, respectively.
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Identification of Functional Protein Regions Through Chimeric Protein Construction
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High coenzyme affinity chimeric amine dehydrogenase based on domain engineering.

Jialin Li1,2,3, Xiaoqing Mu4,5,6, Tao Wu1,2

  • 1Laboratory of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.

Bioresources and Bioprocessing
|April 22, 2024
PubMed
Summary

Engineered chimeric amine dehydrogenase (cFLF-AmDH) enhances NADH cofactor affinity and catalytic efficiency for aromatic chiral amine synthesis. This domain-shuffled enzyme offers improved stability and broader substrate scope for industrial applications.

Keywords:
Amine dehydrogenaseCatalytic efficiencyCoenzyme affinityCoenzyme binding domain

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Area of Science:

  • Biocatalysis and Enzyme Engineering
  • Protein Engineering and Design
  • Synthetic Chemistry

Background:

  • NADH-dependent phenylalanine amine dehydrogenase (F-AmDH) synthesizes aromatic chiral amines but suffers from low coenzyme affinity and catalytic efficiency.
  • Limited industrial applicability of F-AmDH necessitates improvements in enzyme performance.

Purpose of the Study:

  • To develop a chimeric amine dehydrogenase (cFLF-AmDH) with enhanced NADH affinity and catalytic efficiency.
  • To improve the thermal stability and substrate spectrum of F-AmDH through domain shuffling.

Main Methods:

  • Constructed a chimeric enzyme (cFLF-AmDH) by combining substrate-binding domain from F-AmDH and cofactor-binding domain from leucine amine dehydrogenase (L-AmDH).
  • Performed kinetic analyses to evaluate NADH affinity and catalytic efficiency (kcat/Km).
  • Assessed thermal stability and substrate spectrum.
  • Utilized molecular dynamics simulations to understand structural stability and coenzyme binding.

Main Results:

  • cFLF-AmDH exhibited twofold improved NADH affinity and 4.4-fold higher catalytic efficiency compared to parent F-AmDH.
  • Enhanced thermal stability with a 60% longer half-life at 55 °C and a broader substrate spectrum were observed.
  • Molecular dynamics simulations indicated increased structural stability in cFLF-AmDH.
  • Reaction rates for reductive amination increased by 150%, with a 150% conversion rate increase at 0.05 mM NAD+ concentration.

Conclusions:

  • Domain shuffling is an effective strategy for creating enzymes with enhanced cofactor affinity, catalytic efficiency, specificity, and thermal stability.
  • The developed cFLF-AmDH demonstrates significant improvements, making it a promising biocatalyst for industrial applications.
  • This study highlights domain engineering as a viable approach for generating enzyme diversity with tailored catalytic properties.