A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype.

We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Enzyme Immobilization in Wall-Coated Flow Microreactors.

Flow microreactors are emergent engineering tools for the development of continuous biocatalytic transformations. Exploiting enzymes in continuous mode requires their retention for multiple rounds of conversions. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach. However, protein immobilization within closed structures is difficult. Here, we describe a methodology based on the confluent design of enzyme and microreactor; fusion to the silica-binding module Zbasic2 is used to engineer enzymes for high-affinity-oriented attachment to the plain wall surface of glass microchannels. As a practical case, the methodology is described using a sucrose phosphorylase; the assayed reaction is synthesis of α-D-glucose 1-phosphate (αGlc 1-P) from sucrose and phosphate using the immobilized enzyme microreactor. Procedures of enzyme immobilization, reactor characterization, and operation are described. The methodology is applicable for any other enzymes fused to Zbasic2 and silica (glass)-based microfluidic reactors.

Demystifying the Flow: Biocatalytic Reaction Intensification in Microstructured Enzyme Reactors.

Continuous (flow) reactors have drawn a wave of renewed interest in biocatalysis. Many studies find that the flow reactor offers enhanced conversion efficiency. What the reported reaction intensification actually consists in, however, often remains obscure. Here, a canonical microreactor design for heterogeneously catalyzed continuous biotransformations, featuring flow microchannels that contain the enzyme immobilized on their wall surface are examined. Glycosylations by sucrose phosphorylase are used to assess the potential for reaction intensification due to microscale effects. Key variables are identified, and their corresponding relationship equations, to describe, and optimize, the interplay between reaction characteristics, microchannel geometry and reactor operation. The maximum space-time-yield (STY_max) scales directly with the enzyme activity immobilized on the available wall surface. Timescale analysis, comparing the characteristic times of reaction (τreac ) and diffusion (τdiff ) to the mean residence time (τres ), reveals operational conditions for optimum reactor output. Theoretical insight into determinants of microreactor performance is applied to biocatalytic syntheses of α-d-glucose 1-phosphate and α-glucosyl glycerol. Process boundaries for enzyme showing, respectively, high (80 U mg-1 ) and low (4 U mg-1 ) specific activities are thus established and options for process design revealed. Opportunities, and limitations, of the application of principles of microscale flow chemistry to biocatalytic transformations are made evident.

A tailor-made, self-sufficient and recyclable monooxygenase catalyst based on coimmobilized cytochrome P450 BM3 and glucose dehydrogenase.

Cytochrome P450 monooxygenases (P450s) promote hydroxylations in a broad variety of substrates. Their prowess in C-H bond functionalization renders P450s promising catalysts for organic synthesis. However, operating P450 reactions involve complex management of the main substrates, O2 and nicotinamide adenine dinucleotide phosphate (NAD(P)H) reducing equivalents against an overall background of low operational stability. Whole-cell biocatalysis, although often used, offers no general solution to these problems. Herein, we present the design of a tailor-made, self-sufficient, operationally stabilized and recyclable P450 catalyst on porous solid support. Using enzymes as fusion proteins with the polycationic binding module Zbasic2 , the P450s BM3 (from Bacillus megaterium) was coimmobilized with glucose dehydrogenase (type IV; from B. megaterium) on anionic sulfopropyl-activated carrier (ReliSorb SP). Immobilization via Zbasic2 enabled each enzyme to be loaded in controllable amount, thus maximizing the relative portion of the rate limiting P450 BM3 (up to 19.5 U/gcarrier ) in total enzyme immobilized. Using lauric acid as a representative P450 substrate that is poorly accessible to whole-cell catalysts, we demonstrate complete hydroxylation at low catalyst loading (≤0.1 mol%) and efficient electron coupling (74%), inside of the catalyst particle, to the regeneration of NADPH from glucose (27 cycles) was achieved. The immobilized P450 BM3 showed a total turnover number of ∼18,000, thus allowing active catalyst to be recycled up to 20 times. This study therefore supports the idea of practical heterogeneous catalysis by P450s systems immobilized on solid support.

A Spring in Performance: Silica Nanosprings Boost Enzyme Immobilization in Microfluidic Channels.

Enzyme microreactors are important tools of miniaturized analytics and have promising applications in continuous biomanufacturing. A fundamental problem of their design is that plain microchannels without extensive static internals, or packings, offer limited exposed surface area for immobilizing the enzyme. To boost the immobilization in a manner broadly applicable to enzymes, we coated borosilicate microchannels with silica nanosprings and attached the enzyme, sucrose phosphorylase, via a silica-binding module genetically fused to it. We showed with confocal fluorescence microscopy that the enzyme was able to penetrate the ∼70 µm-thick nanospring layer and became distributed uniformly in it. Compared with the plain surface, the activity of immobilized enzyme was enhanced 4.5-fold upon surface coating with nanosprings and further increased up to 10-fold by modifying the surface of the nanosprings with sulfonate groups. Operational stability during continuous-flow biocatalytic synthesis of α-glucose 1-phosphate was improved by a factor of 11 when the microreactor coated with nanosprings was used. More than 85% of the initial conversion rate was retained after 840 reactor cycles performed with a single loading of enzyme. By varying the substrate flow rate, the microreactor performance was conveniently switched between steady states of quantitative product yield (50 mM) and optimum productivity (19 mM min-1) at a lower product yield of 40%. Surface coating with silica nanosprings thus extends the possibilities for enzyme immobilization in microchannels. It effectively boosts the biocatalytic function of a microstructured reactor limited otherwise by the solid surface available for immobilizing the enzyme.