Romina Fernández Varela  Dr. Ana Laura Valino  Dr. Eman Abdelraheem  Dr. Rosario Médici  Dr. Melisa Sayé  Dr. Claudio A. Pereira  Dr. Peter-Leon Hagedoorn  Prof. Ulf Hanefeld  Prof. Adolfo Iribarren  Prof. Elizabeth Lewkowicz 《Chembiochem : a European journal of chemical biology》2022,23(13):e202200147

In nature 2-deoxy-D-ribose-5-phosphate aldolase (DERA) catalyses the reversible formation of 2-deoxyribose 5-phosphate from D-glyceraldehyde 3-phosphate and acetaldehyde. In addition, this enzyme can use acetaldehyde as the sole substrate, resulting in a tandem aldol reaction, yielding 2,4,6-trideoxy-D-erythro-hexapyranose, which spontaneously cyclizes. This reaction is very useful for the synthesis of the side chain of statin-type drugs used to decrease cholesterol levels in blood. One of the main challenges in the use of DERA in industrial processes, where high substrate loads are needed to achieve the desired productivity, is its inactivation by high acetaldehyde concentration. In this work, the utility of different variants of Pectobacterium atrosepticum DERA (PaDERA) as whole cell biocatalysts to synthesize 2-deoxyribose 5-phosphate and 2,4,6-trideoxy-D-erythro-hexapyranose was analysed. Under optimized conditions, E. coli BL21 (PaDERA C-His AA C49M) whole cells yields 99 % of both products. Furthermore, this enzyme is able to tolerate 500 mM acetaldehyde in a whole-cell experiment which makes it suitable for industrial applications.  相似文献   

    

Claudius Lenz  Sebastian Dörner  Felix Trottmann  Prof. Dr. Christian Hertweck  Alexander Sherwood  Prof. Dr. Dirk Hoffmeister 《Chembiochem : a European journal of chemical biology》2022,23(13):e202200183

Psilocybin ( 1 ) is the major alkaloid found in psychedelic mushrooms and acts as a prodrug to psilocin ( 2 , 4-hydroxy-N,N-dimethyltryptamine), a potent psychedelic that exerts remarkable alteration of human consciousness. In contrast, the positional isomer bufotenin ( 7 , 5-hydroxy-N,N-dimethyltryptamine) differs significantly in its reported pharmacology. A series of experiments was designed to explore chemical differences between 2 and 7 and specifically to test the hypothesis that the C-4 hydroxy group of 2 significantly influences the observed physical and chemical properties through pseudo-ring formation via an intramolecular hydrogen bond (IMHB). NMR spectroscopy, accompanied by quantum chemical calculations, was employed to compare hydrogen bond behavior in 4- and 5-hydroxylated tryptamines. The results provide evidence for a pseudo-ring in 2 and that sidechain/hydroxyl interactions in 4-hydroxytryptamines influence their oxidation kinetics. We conclude that the propensity to form IMHBs leads to a higher number of uncharged species that easily cross the blood-brain barrier, compared to 7 and other 5-hydroxytryptamines, which cannot form IMHBs. Our work helps understand a fundamental aspect of the pharmacology of 2 and should support efforts to introduce it (via the prodrug 1 ) as an urgently needed therapeutic against major depressive disorder.  相似文献   

    

Jan Tesarik  Raquel Mendoza-Tesarik 《International journal of molecular sciences》2022,23(23)

Human infertility, of both male and female origin, is often caused by the deficient response of the testis and the ovary to hormonal stimuli that govern sperm and oocyte development and fertilization. The effects of hormones and other extracellular ligands involved in these events are often mediated by G-protein-coupled receptors that employ cyclic adenosine monophosphate (cAMP) as the principal second messenger transducing the receptor-generated signal to downstream elements. This opinion article summarizes the actions of cAMP in sperm and oocyte development and fertilization, leading to therapeutic actions targeting cAMP metabolism to alleviate human male and female infertility.  相似文献   

    

Philipp Wolf  Alexander Mohr  Georgina Gavins  Victoria Behr  Karin Mörl  Prof. Oliver Seitz  Prof. Annette G. Beck-Sickinger 《Chembiochem : a European journal of chemical biology》2022,23(6):e202100340

Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ET_AR and ET_BR), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ET_AR/ET_BR. Further, we demonstrate, that ET_AR expression regulates the signaling of co-expressed ET_BR. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.  相似文献   

    

Hiroshi Takagi  Kohei Kozuka  Kenta Mimura  Prof. Dr. Shogo Nakano  Prof. Dr. Sohei Ito 《Chembiochem : a European journal of chemical biology》2022,23(8):e202100447

Glutamate decarboxylase (GAD) catalyses the decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Improvement of the enzymatic properties of GAD is important for the low-cost synthesis of GABA. In this study, utilizing sequences of enzymes homologous with GAD from lactic acid bacteria, highly mutated GADs were designed using sequence-based protein design methods. Two mutated GADs, FcGAD and AncGAD, generated by full-consensus design and ancestral sequence reconstruction, had more desirable properties than native GADs. With respect to thermal stability, the half-life of the designed GADs was about 10 °C higher than that of native GAD. The productivity of FcGAD was considerably higher than those of known GADs; more than 250 mg/L of purified enzyme could be produced in the E. coli expression system. In a production test using 26.4 g of l -glutamate and 3.0 g of resting cells, 17.2 g of GABA could be prepared within one hour, without purification, in a one-pot synthesis.  相似文献   

    

Zi-Xuan Hu  Cheng Cheng  Yu-Qian Li  Xiao-Han Qi  Dr. Ting Wang  Prof. Dr. Li Liu  Prof. Dr. Josef Voglmeir 《Chembiochem : a European journal of chemical biology》2022,23(13):e202200074

Aldolases are enzymes that reversibly catalyze the cleavage of carbon-carbon bonds. Here we describe a recombinant sialic acid aldolase originating from the freshwater snail Biomphalaria glabrata (sNPL), and compare its substrate spectrum with a sialic acid aldolase originating from chicken (chNPL). In contrast to vertebrate animals which can synthesize, degrade, and incorporate sialic acids on glycoconjugate ubiquitously, snails (as all mollusks) cannot synthesize sialic acids endogenously, and therefore the biological function and substrate scope of sNPL ought to differ significantly from vertebrate sialic aldolases such as chNPL. sNPL was active towards a series of sialic acid derivatives but was in contrast to chNPL unable to catalyze the cleavage of N-acetylneuraminic acid into N-acetylmannosamine and pyruvate. Interestingly, chNPL and sNPL showed contrasting C4(R)/(S) diastereoselectivity towards the substrates d -mannose and d -galactose in the presence of pyruvate. In addition, sNPL was able to synthesize a series of 4-hydroxy-2-oxoates using the corresponding aliphatic aldehyde substrates in the presence of pyruvate, which could be not achieved by chNPL.  相似文献   

    

Dr. Rachel S. Heath  Jack J. Sangster  Prof. Nicholas J. Turner 《Chembiochem : a European journal of chemical biology》2022,23(7):e202200075

The enantioselective oxidation of 2° alcohols to ketones is an important reaction in synthetic chemistry, especially if it can be achieved using O₂-driven alcohol oxidases under mild reaction conditions. However to date, oxidation of secondary alcohols using alcohol oxidases has focused on activated benzylic or allylic substrates, with unactivated secondary alcohols showing poor activity. Here we show that cholesterol oxidase (EC 1.1.3.6) could be engineered for activity towards a range of aliphatic, cyclic, acyclic, allylic and benzylic secondary alcohols. Additionally, since the variants demonstrated high (S)-selectivity, deracemisation reactions were performed in the presence of ammonia borane to obtain enantiopure (R)-alcohols.  相似文献   

    

Dr. Nicole G. H. Leferink  Dr. Andrés M. Escorcia  Bodi R. Ouwersloot  Dr. Linus O. Johanissen  Prof. Dr. Sam Hay  Dr. Marc W. van der Kamp  Prof. Dr. Nigel S. Scrutton 《Chembiochem : a European journal of chemical biology》2022,23(5):e202100688

Monoterpene synthases are often promiscuous enzymes, yielding product mixtures rather than pure compounds due to the nature of the branched reaction mechanism involving reactive carbocations. Two previously identified bacterial monoterpene synthases, a linalool synthase (bLinS) and a cineole synthase (bCinS), produce nearly pure linalool and cineole from geranyl diphosphate, respectively. We used a combined experimental and computational approach to identify critical residues involved in bacterial monoterpenoid synthesis. Phe77 is essential for bCinS activity, guiding the linear carbocation intermediate towards the formation of the cyclic α-terpinyl intermediate; removal of the aromatic ring results in variants that produce acyclic products only. Computational chemistry confirmed the importance of Phe77 in carbocation stabilisation. Phe74, Phe78 and Phe179 are involved in maintaining the active site shape in bCinS without a specific role for the aromatic ring. Phe295 in bLinS, and the equivalent Ala301 in bCinS, are essential for linalool and cineole formation, respectively. Where Phe295 places steric constraints on the carbocation intermediates, Ala301 is essential for bCinS initial cyclisation and activity. Our multidisciplinary approach gives unique insights into how carefully placed amino acid residues in the active site can direct carbocations down specific paths, by placing steric constraints or offering stabilisation via cation-π interactions.  相似文献   

    

Dr. Laura Drummond  Parab J. Haque  Dr. Binbin Gu  Julia S. Jung  Dr. Hendrik Schewe  Prof. Dr. Jeroen S. Dickschat  Dr. Markus Buchhaupt 《Chembiochem : a European journal of chemical biology》2022,23(14):e202200091

The natural substance class of terpenoids covers an extremely wide range of different structures, although their building block repertoire is limited to the C₅ compounds DMAPP and IPP. This study aims at the characterization of methyltransferases (MTases) that modify these terpene precursors and the demonstration of their suitability for biotechnological purposes. All seven enzymes tested accepted IPP as substrate and altogether five C₆ compounds and six C₇ compounds were formed within the reactions. A high selectivity for the deprotonation site as well as high stereoselectivity could be observed for most of the biocatalysts. Only the enzyme from Micromonospora humi also accepted DMAPP as substrate, converting it into (2R)-2-methyl-IPP in vitro. In vivo studies demonstrated the production of a C₈ compound and a hydride shift step within the MTase-catalyzed reaction. Our study presents IPP/DMAPP MTases with very different catalytic properties, which provide biosynthetic access to many novel terpene-derived structures.  相似文献   

    

Yannik Brack  Dr. Chenghai Sun  Prof. Dr. Dong Yi  Prof. Dr. Uwe T. Bornscheuer 《Chembiochem : a European journal of chemical biology》2022,23(10):e202200062

p-Coumaric acid (p-CA) is a key precursor for the biosynthesis of flavonoids. Tyrosine ammonia lyases (TALs) specifically catalyze the synthesis of p-CA from l -tyrosine, which is a convenient enzymatic pathway. To explore novel and highly active TALs, a phylogenetic tree-building approach was conducted including 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases (PTALs). Among them, 5 TALs and 3 PTALs were successfully characterized and found to exhibit the proposed enzymatic activity. The TAL from Chryseobacterium luteum sp. nov (TAL_clu) has the highest affinity (K_m =0.019 mm ) and conversion efficiency (k_cat/K_m= 1631 s⁻¹ ⋅ mm ⁻¹) towards l -tyrosine. The reaction conditions for two purified enzymes and their E. coli recombinant cells were optimized and p-CA yields of 2.03 g/L after 8 hours by TAL_clu and 2.35 g/L after 24 h by TAL from Rivularia sp. PCC 7116 (TAL_rpc) in whole cells were achieved. These TALs are thus candidates for the construction of whole-cell systems to produce the flavonoid precursor p-CA.  相似文献