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. 相似文献
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. 相似文献
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. 相似文献
The enzyme microreactor has considerable potential for use in biotechnological syntheses and analytical studies. Simplifying the procedure of enzyme immobilization in a microreactor is attractive, and it is achievable by utilizing enzyme immobilization techniques and taking advantage of the characteristics of microfluidics. We previously developed a facile and inexpensive preparation method for an enzyme‐immobilized microreactor. The immobilization of enzymes can be achieved by the formation of an enzyme‐polymeric membrane on the inner wall of the microchannel through cross‐linking polymerization in a laminar flow. However, this method is unsuitable for use in conjunction with electronegative enzymes. Therefore, a novel preparation method using poly‐L ‐lysine [poly(Lys)] as a booster and an adjunct for the effective polymerization of electronegative enzymes was developed in this study. Using aminoacylase as a model for an electronegative enzyme, the reaction conditions for the enzyme‐cross‐linked aggregation were optimized. On the basis of the determined conditions, an acylase‐immobilized tubing microreactor was successfully prepared by cross‐linking polymerization in a concentric laminar flow. The resulting microreactor showed a higher stability against heat and organic solvents compared to those of the free enzyme. The developed method using poly(Lys) was applicable to various enzymes with low isoelectric points, suggesting that this microreactor preparation utilizing a cross‐linked enzyme in a laminar flow could be expanded to microreactors in which a broad range of functional proteins are employed. 相似文献
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. 相似文献
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 Km for (R)‐mandelonitrile and kcat 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. 相似文献
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. 相似文献
The chiral pyrrolidine‐substituted imidazolium cetyl‐PEG10‐sulfate (D ‐ProMe) derived from D ‐proline worked as an excellent activating agent of Burkholderia cepacia lipase; it is particularly interesting that the D ‐isomer of the imidazolium salt worked better than the L ‐isomer. This suggests that the imidazolium cation group directly interacts with the enzyme protein and causes preferable modification of the reactivity. 相似文献
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. 相似文献
A new catalyst for an old material: magnetite is an excellent Lewis acid catalyst for the four‐component aza‐Sakurai reaction. The process could be repeated up to 15‐times without losing effectiveness, with the catalyst recycling being as easy as the use of a simple magnet. The catalyst is selective and could discriminate between aldehyde and ketone functionalities, catalyzing first the reaction with the higher electrophilic aldehyde. 相似文献
Isomaltulose is a new functional sweetener that possesses biological characteristics and can be obtained by enzymatic synthesis using sucrose as substrate. In this study, the sucrose isomerase (SIase) was successfully immobilized on the sponge synthesized with ϵ‐poly‐l ‐lysine and gelatin. The inhibition zones proved that the sponge possessed a significant bacteriostatic function, which can effectively prevent the collapse of the scaffolds of the immobilized enzymes. The immobilized SIase activity reached up to 71.58 U g−1 (the SIase loading was about 153.43 mg g−1), and the activity recovery was approximately 84.50%. After immobilization, the optimum pH of SIase decreased from 6.0 to 5.5 and the optimum temperature increased from 30 °C to 40 °C. The affinity of SIase to substrate was basically unchanged. Immobilized SIase still exhibited more than 90% sucrose conversion after 13 consecutive cycles, which indicated that it had a good operational stability. Furthermore, the immobilized SIase has the potential for isomaltulose production, with 200 g L−1 sucrose solution as its substrate in the food industry. Isomaltulose was isolated in 83.58% yield and high purity (97.3%).
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. 相似文献