A New Thiolate-Bound Dimanganese Cluster as a Structural and Functional Model for Class Ib Ribonucleotide Reductases

In class Ib ribonucleotide reductases (RNRs) a dimanganese(II) cluster activates superoxide (O₂⋅⁻) rather than dioxygen (O₂), to access a high valent Mn^III−O₂−Mn^IV species, responsible for the oxidation of tyrosine to tyrosyl radical. In a biomimetic approach, we report the synthesis of a thiolate-bound dimanganese complex [Mn^II₂(BPMT)(OAc)₂](ClO)₄ (BPMT=(2,6-bis{[bis(2-pyridylmethyl)amino]methyl}-4-methylthiophenolate) ( 1 ) and its reaction with O₂⋅⁻ to form a [(BPMT)MnO₂Mn]²⁺ complex 2 . Resonance Raman investigation revealed the presence of an O−O bond in 2 , while EPR analysis displayed a 16-line S_t=1/2 signal at g=2 typically associated with a Mn^IIIMn^IV core, as detected in class Ib RNRs. Unlike all other previously reported Mn−O₂−Mn complexes, generated by O₂⋅⁻ activation at Mn₂ centers, 2 proved to be a capable electrophilic oxidant in aldehyde deformylation and phenol oxidation reactions, rendering it one of the best structural and functional models for class Ib RNRs.     

Recent developments in oxidative biocatalytic cascades

Biocatalytic cascades that involve enzymatic oxidation as one or more key steps are powerful tools to access valuable chemicals with various functionalizations starting from simple substrates, without the isolation of intermediates. This review discusses the recent advances in oxidative cascades, with perspectives given on the current limitations and future developments. The strategies employed to achieve efficient supply of redox cofactors are also highlighted. The examples include cascades that begin with alkene epoxidation, alkane hydroxylation, alcohol oxidation or amine oxidation. These oxidative steps are followed by a variety of enzyme-catalyzed functionalizations, producing a diverse range of high-value products.     

Aziridination of γ,δ-dibromoethyl-2-pentenoate with primary amines: extension of the Gabriel-Cromwell reaction

Ethyl (E)-4,5-dibromo-2-pentenoate readily reacts with an assortment of primary amines in the presence of DBU to afford the corresponding conjugated aziridines in good to moderate yields. That the reaction is compatible with a nucleoside-derived amine suggests a broad scope of application.     

Bioelectrocatalytic CO2 Reduction by Mo-Dependent Formylmethanofuran Dehydrogenase

Massive efforts are invested in developing innovative CO₂-sequestration strategies to counter climate change and transform CO₂ into higher-value products. CO₂-capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO₂, it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO₂-reducing-and-fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO₂ reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO₂-fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.     

Biocatalytic reduction of prochiral aromatic ketones to optically pure alcohols by a coupled enzyme system for cofactor regeneration

A simple, highly efficient, and economical biphasic cell-free system was developed for biocatalytic reduction of prochiral aromatic ketones to furnish enantiopure alcohols. This system is characterized by using endogenous enzymes of the cell-free extract to form enzyme-coupled NADPH recycling system. Besides, it offered much higher productivity than whole cells and greatly simplified the preparation process of biocatalysts in comparison with isolated enzymes. Various prochiral aromatic ketones, especially α-substituted acetophenone derivatives, were reduced to chiral alcohols with excellent enantiomeric excess (ee) and moderate to good yield by this cell-free system.     

基于辅因子化学本质及功能的分类与讨论

辅因子是蛋白质或酶结构的重要组成部分,在许多酶的功能中发挥重要乃至关键作用。在科研和教学中,现有的辅因子概念及其分类系统不够清晰准确。本文从化学本质和功能上将辅因子分为催化型、载体型和底物型3类,并以若干典型的辅因子为例讨论辅因子的特异性及再生性等问题。     

Mutation of Tyr-218 to Phe in <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP⁺), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD⁺). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD⁺ by mutating Tyr²¹⁸ to Phe (Y218F 2° ADH). This mutation increased the K _m(app) for NADP⁺ 200-fold while decreasing the K _m(app) for NAD⁺ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD⁺, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD⁺. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD⁺ was electrically recycled 6.7 × 10⁴ times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.     

Synthesis and Structure of Vitamin B<Subscript>12</Subscript>-Derivatives with a Modified Ribose-Unit

Summary. The acylation at the 5′-OH group of the ribose-unit of vitamin B₁₂ (cyanocobalamin) or of aquocobalamin with two conventional reagents gave mono-acylated B₁₂-derivatives with good to very high selectivity. The site of the modification was deduced from spectral data of the products and was further supported by the crystal structure data of three such modified B₁₂-derivatives. These three B₁₂-derivatives were found to crystallize in the space group P2₁2₁2₁, irrespective of the nature of the appendage. Acylation at 5′-OH has been used to protect (or block) this group in the context of functionalization of 2′-OH or elsewhere in the B₁₂-molecule. Attachment of the bifunctional succinyl-unit has allowed the preparation of further modified derivatives of vitamin B₁₂ and binding of B₁₂-derivatives to biological carriers and other macromolecules. In aqueous solution, 5′-acylcobalamins turned out to be rather susceptible to hydrolytic loss of the acyl-functionality. Bernhard Kr?utler: In memoriam Prof. Karl Schl?gl