A Designed Enzyme Promotes Selective Post‐translational Acylation

A computationally designed, allosterically regulated catalyst (CaM M144H) produced by substituting a single residue in calmodulin, a non‐enzymatic protein, is capable of efficient and site selective post‐translational acylation of lysines in peptides with highly diverse sequences. Calmodulin′s binding partners are involved in regulating a large number of cellular processes; this new chemical‐biology tool will help to identify them and provide structural insight into their interactions with calmodulin.     

A Membrane‐Bound Prenyltransferase Catalyzes the O‐Prenylation of 1,6‐Dihydroxyphenazine in the Marine Bacterium Streptomyces sp. CNQ‐509

Streptomyces sp. CNQ‐509 produces the rare O‐prenylated phenazines marinophenazines A and B. To identify the enzyme catalyzing the O‐prenyl transfer in marinophenazine biosynthesis, we sequenced the genome of S. sp. CNQ‐509. This led to the identification of two genomic loci harboring putative phenazine biosynthesis genes. The first locus contains orthologues for all seven genes involved in phenazine‐1‐carboxylic acid biosynthesis in pseudomonads. The second locus contains two known phenazine biosynthesis genes and a putative prenyltransferase gene termed cnqPT1. cnqPT1 codes for a membrane protein with sequence similarity to the prenyltransferase UbiA of ubiquinone biosynthesis. The enzyme CnqPT1 was identified as a 1,6‐dihydroxyphenazine geranyltransferase, which catalyzes the C?O bond formation between C‐1 of the geranyl moiety and O‐6 of the phenazine scaffold. CnqPT1 is the first example of a prenyltransferase catalyzing O‐prenyl transfer to a phenazine.     

Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction

An enzyme that catalyzes the formose reaction, termed “formolase”, was recently engineered through a combination of computational protein design and directed evolution. We have investigated the kinetic role of the computationally designed residues and further characterized the enzyme's product profile. Kinetic studies illustrated that the computationally designed mutations were synergistic in their contributions towards enhancing activity. Mass spectrometry revealed that the engineered enzyme produces two products of the formose reaction—dihydroxyacetone and glycolaldehyde—with the product profile dependent on the formaldehyde concentration. We further explored the effects of this product profile on the thermodynamics and yield of the overall carbon assimilation from the formolase pathway to help guide future efforts to engineer this pathway.     

Mutagenesis of an Asn156 Residue in a Surface Region of S‐Selective Hydroxynitrile Lyase from Baliospermum montanum Enhances Catalytic Efficiency and Enantioselectivity

The S‐selective hydroxynitrile lyase from Baliospermum montanum (BmHNL) has broad substrate specificity toward aromatic substrates as well as high temperature stability, although with low enantioselectivity and specific activity. To expand the industrial application of this enzyme, we improved its enantioselectivity and specific activity toward (S)‐mandelonitrile by mutagenesis. The specific activity of the BmHNL H103C/N156G mutant for (S)‐mandelonitrile production was raised to 154 U mg^?1 (WT BmHNL: 52 U mg^?1). The enantiomeric excess was increased to 93 % (WT BmHNL: 55 %). The kinetic analysis revealed K_m for (R)‐mandelonitrile and k_cat for (S)‐mandelonitrile increased by the mutation at Asn156, thus contributing to the increase in enantiomeric excess. This is the first report on an improvement in catalytic efficiency and enantiomeric excess of BmHNL for (S)‐mandelonitrile synthesis by random and site‐directed mutagenesis.     

Single‐Molecule Kinetics of an Enzyme in the Presence of Multiple Substrates

Herein, the catalytic activity of a single enzyme in the presence of multiple substrates is studied. Three different mechanisms of bisubstrate binding, namely, ordered sequential, random sequential and ping‐pong nonsequential pathway, are broadly discussed. By means of the chemical master equation approach, exact expressions for the waiting‐time distributions, the mean turnover time and the randomness parameter as a function of the substrate concentration, such that both concentrations are fixed, but one of them is changed quasi‐statically are obtained. The randomness parameter is not equal to unity at intermediate to high substrate concentrations, which indicates the presence of multiple rate‐limiting steps in the reaction pathway in all three modes of bisubstrate binding. This arises due to transitions between the free enzyme and the enzyme–substrate complexes that occur on comparable timescales. Such turnover statistics of the single enzyme can also distinguish between the different types of bisubstrate binding mechanisms.     

Removing the Active‐Site Flap in Lipase A from Candida antarctica Produces a Functional Enzyme without Interfacial Activation

A mobile region is proposed to be a flap that covers the active site of Candida antarctica lipase A. Removal of the mobile region retains the functional properties of the enzyme. Interestingly interfacial activation, required for the wild‐type enzyme, was not observed for the truncated variant, although stability, activity, and stereoselectivity were very similar for the wild‐type and variant enzymes. The variant followed classical Michaelis–Menten kinetics, unlike the wild type. Both gave the same relative specificity in the transacylation of a primary and a secondary alcohol in organic solvent. Furthermore, both showed the same enantioselectivity in transacylation of alcohols and the hydrolysis of alcohol esters, as well as in the hydrolysis of esters chiral at the acid part.     

Aqueous Oxidative Heck Reaction as a Protein‐Labeling Strategy

An increasing number of chemical reactions are being employed for bio‐orthogonal ligation of detection labels to protein‐bound functional groups. Several of these strategies, however, are limited in their application to pure proteins and are ineffective in complex biological samples such as cell lysates. Here we present the palladium‐catalyzed oxidative Heck reaction as a new and robust bio‐orthogonal strategy for linking functionalized arylboronic acids to protein‐bound alkenes in high yields and with excellent chemoselectivity even in the presence of complex protein mixtures from living cells. Advantageously, this reaction proceeds under aerobic conditions, whereas most other metal‐catalyzed reactions require inert atmosphere.     

Site‐Specific Modification of the 6‐Amino Group of Adenosine in RNA by an Interstrand Functionality‐Transfer Reaction With an S‐Functionalized 4‐Thiothymidine

Non‐natural RNA modifications have been widely used to study the function and structure of RNA. Expanding the study of RNA further requires versatile and efficient tools for site‐specific RNA modification. We recently established a new strategy for the site‐specific modification of RNA based on a functionality‐transfer reaction between an oligodeoxynucleotide (ODN) probe and an RNA substrate. 2′‐Deoxy‐6‐thioguanosine was used to anchor the transfer group, and the 4‐amino group of cytosine or the 2‐amino group of guanine was specifically modified. In this study, 2′‐deoxy‐4‐thiothymidine was adopted as a new platform to target the 6‐amino group of adenosine. The (E)‐pyridinyl vinyl keto transfer group was attached to the 4‐thioT in the ODN probe, and it was efficiently and specifically transferred to the 6‐amino group of the opposing adenosine in RNA in the presence of CuCl₂. This method expands the available RNA target sites for specific modification.     

Tailored Synthesis of 162‐Residue S‐Monoglycosylated GM2‐Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library

A synthetic protocol for the preparation of 162‐residue S‐monoglycosylated GM2‐activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one‐pot/N‐to‐C‐directed sequential ligation strategy using readily accessible middle N‐sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1–31)] with the N‐terminal cysteinyl prolyl thioester [GM2AP (32–67)] proceeded smoothly to yield the 67‐residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as “tailored synthesis” for the construction of a GM2AP library.     

Microbial Transglutaminase and c‐myc‐Tag: A Strong Couple for the Functionalization of Antibody‐Like Protein Scaffolds from Discovery Platforms

Antibody‐like proteins selected from discovery platforms are preferentially functionalized by site‐specific modification as this approach preserves the binding abilities and allows a side‐by‐side comparison of multiple conjugates. Here we present an enzymatic bioconjugation platform that targets the c‐myc‐tag peptide sequence (EQKLISEEDL) as a handle for the site‐specific modification of antibody‐like proteins. Microbial transglutaminase (MTGase) was exploited to form a stable isopeptide bond between the glutamine on the c‐myc‐tag and various primary‐amine‐functionalized substrates. We attached eight different functionalities to a c‐myc‐tagged antibody fragment and used these bioconjugates for downstream applications such as protein multimerization, immobilization on surfaces, fluorescence microscopy, fluorescence‐activated cell sorting, and in vivo nuclear imaging. The results demonstrate the versatility of our conjugation strategy for transforming a c‐myc‐tagged protein into any desired probe.     

Phenylalanine Ammonia‐Lyase‐Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

Phenylalanine ammonia‐lyase (PAL), found in many organisms, catalyzes the deamination of l ‐phenylalanine (Phe) to (E)‐cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in‐line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)‐pent‐2‐ene‐4‐ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts‐type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)‐pent‐2‐ene‐4‐ynoate yielding enantiopure l ‐PG, contradicts the proposed highly exothermic single‐step mechanism. Computations with the QM/MM models of the N‐MIO intermediates from l ‐PG and l ‐Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N‐MIO intermediate.     

An Engineered Old Yellow Enzyme that Enables Efficient Synthesis of (4R,6R)‐Actinol in a One‐Pot Reduction System

(4R,6R)‐Actinol can be stereo‐selectively synthesized from ketoisophorone by a two‐step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)‐levodione reductase. However, (4S)‐phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p‐HBA, and introduced point mutations into the substrate‐recognition loop. The most effective mutant (P295G) showed two‐ and 12‐fold higher catalytic activities toward ketoisophorone and (4S)‐phorenol, respectively, than the wild‐type, and improved the yield of the two‐step conversion from 67.2 to 90.1 %. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate‐recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.