Volker Brecht

Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway.

Fifty years ago, Kornberg and Krebs established the glyoxylate cycle as the pathway for the synthesis of cell constituents from C2-units. However, since then, many bacteria have been described that do not contain isocitrate lyase, the key enzyme of this pathway. Here, a pathway termed the ethylmalonyl-CoA pathway operating in such organisms is described. Isotopically labeled acetate and bicarbonate were transformed to ethylmalonyl-CoA by cell extracts of acetate-grown, isocitrate lyase-negative Rhodobacter sphaeroides as determined by NMR spectroscopy. Crotonyl-CoA carboxylase/reductase, catalyzing crotonyl-CoA + CO2 + NADPH → ethylmalonyl-CoA− + NADP+ was identified as the key enzyme of the ethylmalonyl-CoA pathway. The reductive carboxylation of an enoyl-thioester is a unique biochemical reaction, unprecedented in biology. The enzyme from R. sphaeroides was heterologously produced in Escherichia coli and characterized. Crotonyl-CoA carboxylase/reductase (or its gene) can be used as a marker for the presence of the ethylmalonyl-CoA pathway, which functions not only in acetyl-CoA assimilation. In Streptomyces sp., it may also supply precursors (ethylmalonyl-CoA) for antibiotic biosynthesis. For methylotrophic bacteria such as Methylobacterium extorquens, extension of the serine cycle with reactions of the ethylmalonyl-CoA pathway leads to a simplified scheme for isocitrate lyase-independent C1 assimilation.

Identifying the missing steps of the autotrophic 3-hydroxypropionate CO2 fixation cycle in Chloroflexus aurantiacus

The phototrophic bacterium Chloroflexus aurantiacus uses a yet unsolved 3-hydroxypropionate cycle for autotrophic CO2 fixation. It starts from acetyl-CoA, with acetyl-CoA and propionyl-CoA carboxylases acting as carboxylating enzymes. In a first cycle, (S)-malyl-CoA is formed from acetyl-CoA and 2 molecules of bicarbonate. (S)-Malyl-CoA cleavage releases the CO2 fixation product glyoxylate and regenerates the starting molecule acetyl-CoA. Here we complete the missing steps devoted to glyoxylate assimilation. In a second cycle, glyoxylate is combined with propionyl-CoA, an intermediate of the first cycle, to form β-methylmalyl-CoA. This condensation is followed by dehydration to mesaconyl-C1-CoA. An unprecedented CoA transferase catalyzes the intramolecular transfer of the CoA moiety to the C4 carboxyl group of mesaconate. Mesaconyl-C4-CoA then is hydrated by an enoyl-CoA hydratase to (S)-citramalyl-CoA. (S)-Citramalyl-CoA is cleaved into acetyl-CoA and pyruvate by a tri-functional lyase, which previously cleaved (S)-malyl-CoA and formed β-methylmalyl-CoA. Thus, the enigmatic disproportionation of glyoxylate and propionyl-CoA into acetyl-CoA and pyruvate is solved in an elegant and economic way requiring only 3 additional enzymes. The whole bicyclic pathway results in pyruvate formation from 3 molecules of bicarbonate and involves 19 steps but only 13 enzymes. Elements of the 3-hydroxypropionate cycle may be used for the assimilation of small organic molecules. The 3-hydroxypropionate cycle is compared with the Calvin–Benson–Bassham cycle and other autotrophic pathways.

Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase

Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO2. Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis.

Parameters influencing the determination of liposome lamellarity by 31P-NMR.

The lamellarity of liposomes influences to a great extent the encapsulation efficiency, the efflux rate of liposomally encapsulated material, and the fate of a drug after cellular uptake. 31P-NMR in combination with the use of chemical shift reagents has been described for the determination of lamellarity of liposomes and this study was performed to evaluate the applicability of 31P-NMR analysis as published in the past. To date, very few details about the required conditions throughout the measurements are known. In this study the influence of various parameters on the measurement, such as different buffers with changing ion concentrations, varying pH and different shift reagents at increasing concentrations was investigated. Results were discussed by using cryo-electronmicroscopy as a reference method. The data of this study show that 31P-NMR might not result in the correct determination of liposome lamellarity, depending on the experimental settings and the shape of the liposomes.

Identification of the cannabimimetic AM-1220 and its azepane isomer (N-methylazepan-3-yl)-3-(1-naphthoyl)indole in a research chemical and several herbal mixtures

Recently, a large number of synthetic cannabinoids have been identified in herbal mixtures. Moreover, an even higher number of cannabimimetic compounds are currently distributed as research chemicals on a gram to kilogram scale via several online trading platforms. As this situation leads to a large number of new cannabimimetics and the occurrence of isobaric substances, the analysis of such compounds using mass spectroscopy (MS) involves the risk of incorrect assignments of mass spectra. In certain cases, this leads to considerable analytical challenges. In the majority of cases, these challenges can only be mastered by combining multiple analytical techniques. We purchased a so-called research chemical advertised as the cannabimimetic compound [(N-methylpiperidin-2-yl)methyl]-3-(1-naphthoyl)indole (AM-1220) via an Internet platform. Analysis of the microcrystalline substance using gas chromatography (GC)–MS indicated the presence of pure AM-1220. However, after further purity testing utilizing thin-layer chromatography we were surprised to see an additional spot indicating a mixture of two substances with highly similar physicochemical properties. After isolation, high-resolution mass spectroscopy (HR-MS) revealed an elemental composition of C26H26N2O for both substances, proving the presence of two isobaric substances. Moreover, GC–MS and LC-HR-MS/MS experiments indicated two naphthoylindoles featuring different heterocyclic substituents at the indole nitrogen. Nuclear magnetic resonance spectroscopy verified the presence of the highly potent cannabimimetic AM-1220 and its azepane isomer. Interestingly, only a few weeks after purchasing the powder we also detected both substances in a similar proportion in several herbal mixtures for the first time.

Elucidation of the Enantioselective Cyclohexane‐1,2‐dione Hydrolase Catalyzed Formation of (S)‐Acetoin

Thiamine diphosphate (ThDP) dependent enzymes catalyze the formation of acetoin (3‐hydroxybutan‐2‐one) through one of three different pathways: homocoupling of pyruvate, homocoupling of acetaldehyde, or cross‐coupling of acetaldehyde (as acceptor) and pyruvate (as donor). The enantioselectivity of the resulting acetoin is highly dependent on the particular enzyme. We established that ThDP‐dependent cyclohexane‐1,2‐dione hydrolase (CDH) is able to form (S)‐acetoin with particularly high enantioselectivity (up to 95 % ee) by all three pathways. Mechanistic studies utilizing 13C‐labeled substrates revealed an unprecedented non‐acetolactate pathway for the homocoupling of pyruvate, which explains the high enantioselectivity in the CDH‐catalyzed formation of (S)‐acetoin.

E/Z -Isomerism of semicyclic amidinium salts

1H and 15N NMR chemical shifts of 12 semicyclic amidinium salts are reported. Their E/Z equilibria in solution were analyzed by 1D and 2D 1H and 15N NMR techniques. In the solid state the Z ‐configuration is observed.