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Hock, K. J., Knorrscheidt, A., Hommelsheim, R., Ho, J., Weissenborn, M. J. & Koenigs, R. M. Iron-Catalyzed C—H Insertions: Organometallic and Enzymatic Carbene Transfer Reactions ChemRxiv (2018) DOI: 10.26434/chemrxiv.6011096

C—H insertion reactions with organometallic and enzymatic catalysts based on earth-abundant iron complexes remain one of the major challenges in organic synthesis. In this report, we describe the development and application of these iron-based catalysts in the reaction of two different carbene precursors with N-heterocycles for the first time. While FeTPPCl showed excellent reactivity in the Fe(III) state with diazoacetonitrile, the highest activities of the YfeX enzyme could be achieved upon heme-iron reduction to Fe(II) with both diazoacetonitrile and ethyl diazoacetate. This highlights unexpected and subtle differences in reactivity of both iron catalysts. Deuterium labeling studies indicated a C—H insertion pathway and a marked kinetic isotope effect. This transformation features mild reaction conditions, excellent yields or turnover numbers with broad functional group tolerance, including gram-scale applications giving a unique access to functionalized N-heterocycles.

Darimont, D., Weissenborn, M. J., Nebel, B. A. & Hauer, B. Modulating proposed electron transfer pathways in P450BM3 led to improved activity and coupling efficiency.  Bioelectrochemistry 119, 119-123, (2018) DOI: 10.1016/j.bioelechem.2017.08.009

Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450BM3 variants for aniline hydroxylation, equipped with a carbon nanotube binding-peptide (CNT-tag) for self-assembly on CNT electrodes. Compared to wildtype P450BM3 the NADPH-dependent activity was enhanced, but the coupling efficiency remained low. For P450BM3 Verma, Schwaneberg and Roccatano (2014, Biopolymers 101, 197–209) calculated putative electron transfer pathways (eTPs) by MD simulations. We hypothesised that knockouts of these transfer pathways would alter the coupling efficiency of the system. The results revealed no improved system for the electrically-driven P450s. For the NADPH-driven P450s, however, the most active eTP-mutant showed a 13-fold increased activity and a 32-fold elevated coupling efficiency using NADPH as reducing equivalent. This suggests an alternative principle of electron transport for the reduction by NADPH and an electrode, respectively. The work presents moreover a tool to improve the coupling and activity of P450s with non-natural substrates.

Weissenborn, M. J., Debecker, D. P., Golten, S., Linclau, B., Turner, N. J. & Flitsch, S. L. Development of a Solid Phase Array Assay for the Screening of Galactose Oxidase Activity and for Fast Identification of Inhibitors.  Protein Pept Lett. 24(8), 742-746, (2017) DOI: 10.2174/0929866524666170724114348

Galactose oxidase (GOase) catalyses the highly selective oxidation of terminal galactosides on a wide range of natural glycoconjugates and has found wide applications in biotechnology - particularly in biocatalysis. GOase is copper dependent and uses oxygen to oxidise the C6-primary alcohol of galactose and produces hydrogen peroxide. The enzyme activity can be conveniently assessed by a colorimetric assay.

The objective of the present study was to develop an assay system, which is independent of the hydrogen peroxide formation to identify possible fluorinated GOase inhibitors. In case that the inhibitor bears a primary or secondary alcohol, it could also be oxidised by the enzyme. In such case, the colorimetric assay is not able to distinguish between substrate and inhibitor, since oxidation of both molecules would result in the formation of hydrogen peroxide.

D-galactose (D-Gal) was immobilised onto a gold surface functionalised by selfassembled monolayers (SAMs,). A GOase solution was then added to the surface in a droplet for a certain period of time and thereafter washed away. The activity of GOase on the immobilised D-Gal can then be quantified by MALDI-ToF MS.

For inhibition studies, GOase was incubated together with 62.5 mM of deoxy-fluorinated monosaccharides on the D-Gal displaying platform. Five deoxy-fluorinated D-Gal showed a >50% inhibition of its activity. The array system has been moreover utilised to determine the apparent IC50 value of 3-F-Gal 15 as a proof of principle.

The developed array platform allows the fast identification of GOase substrates and inhibitors from a library of deoxy-fluorinated sugars using MALDI-ToF MS as a label-free readout method. In addition, the enzymatic reaction enables for the in situ activation of sugar-coated surfaces to bioorthogonal aldehydes, which can be utilised for subsequent chemical modifications.


Kammel, M., Knorrscheidt, A., Püllmann, P. & Weissenborn, M. J. Tackling the numbers problem: Entwicklung nicht-nativer Enzymreaktionen.  BIOspektrum 23(7), 830-832, (2017) DOI: 10.1007/s12268-017-0876-3

The screening effort of large protein variant libraries renders the probability of coincidental discovering a new enzyme with non-natural activity to almost zero - the so-called numbers problem. Insights into the origin of life, evolution and enzymatic promiscuity, combined with the inspiration of methods from organic chemistry, offer solutions for this problem. With the newly discovered enzymes synthetic micro production units shall be established in a Leibniz Research Cluster where engineering and biotechnology are combined.

Weissenborn, M. J., Notonier, S., Lang, S. L., Otte, K. B., Herter, S., Turner, N. J., Flitsch, S. L. & Hauer, B. Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s Chem. Commun. 52, 6158-6161, (2016) DOI: 10.1039/C6CC01749E

A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids.


Weissenborn, M. J., Low, S. A., Borlinghaus, N., Kuhn, M., Kummer, S., Rami, F., Plietker, B. & Hauer, B. Enzyme-Catalyzed Carbonyl Olefination by the E. coli Protein YfeX in the Absence of Phosphines ChemCatChem 8, 1636-1640, (2016) DOI: 10.1002/cctc.201600227

The Wittig-type carbonyl olefination reaction has no biocatalytic equivalent. To build complex molecular scaffolds, however, C−C bond-forming reactions are pivotal for biobased economy and synthetic biology. The heme-containing E. coli protein YfeX was found to catalyze carbonyl olefination by reaction of benzaldehyde with ethyl diazoacetate under aerobic conditions in the absence of a triphenylphosphine oxophile. The reaction was performed in whole cells and showed a product formation of 440 mg L−1 in 1 h. It was, moreover, shown that the reaction could be performed under Wittig-analogue conditions in the presence of triphenylphosphine or triphenylarsine.


Low, S. A., Low, I. M., Weissenborn, M. J. & Hauer, B. Enhanced Ene-Reductase Activity through Alteration of Artificial Nicotinamide Cofactor Substituents ChemCatChem 8, 911-915, (2016) DOI: 10.1002/cctc.201501230

The reduction of activated C=C double bonds is an important reaction in synthetic chemistry owing to the potential formation of up to two new stereogenic centers. Artificial nicotinamide cofactors were recently presented as alternative suppliers of hydride equivalents needed for alkene reduction. To study the effect of cofactors on the reduction of activated alkenes, a set of N-substituted synthetic nicotinamide cofactors with differing oxidation potentials were synthesized and their electrochemical and kinetic behavior was studied. The effects of the synthetic cofactors on enzyme activity of four ene reductases are outlined in this study, where the cofactor mimic with an N-substituted 4-hydroxy-phenyl residue led to a sixfold higher vmax relative to the natural cofactor NADH.


Llaudet, E. C., Darimont, D., Samba, R., Matiychyn, I., Stelzle, M., Weissenborn, M. J. & Hauer, B. Expanding an Efficient, Electrically Driven and CNT-Tagged P450 System into the Third Dimension: A Nanowired CNT-Containing and Enzyme-Stabilising 3 D Sol-Gel Electrode. ChemBioChem 17, 1367-1373, (2016) DOI: 10.1002/cbic.201600173

Although electrochemically catalysed P450 reactions have been described, their efficiency and applicability remained limited. This is mostly due to low enzyme activity, laborious protein immobilisation and the small electrode surface. We established a novel protein immobilisation method for a determined orientation and electrical wiring of the enzyme without post-expression modification. By genetic introduction of an anchor-peptide our method is applicable for screening medium to large mutant libraries and detection by an electrode system. The system was expanded by using wired carbon nanotubes within a sol-gel matrix to create a three dimensional electrode.


Hoffmann, S. M., Weissenborn, M. J., Gricman, L., Notonier, S., Pleiss, J. & Hauer, B. The Impact of Linker Length on P450 Fusion Constructs: Activity, Stability and Coupling ChemCatChem 8, 1591-1597, (2016) DOI: 10.1002/cctc.201501397

Three different reductases have been fused to CYP153 monooxygenase from Marinobacter aquaeolei. The most promising candidate has been analysed in terms of its linker part, which connects the reductase with the haem domain through sequence alignment of the corresponding reductase family CYP116B. To improve the artificial fusion construct, the linker length has been varied, thereby only altering the non-conserved middle part of the linker. This way seven artificial fusion constructs have been engineered, which varied in linker length between 11 and 32 amino acids (“natural” is 16). These variations showed a substantial impact on the fusion construct. The best mutant, extended by two amino acids, showed an improved activity (67 %), higher stability (67 % more active haem domain after 2 h) and a coupling efficiency of 94 % (55 % higher than before). Presented in this paper is an approach to find and optimise artificial fusion constructs for P450 monooxygenases.


Hoffmann, S. M., Danesh-Azari, H.-R., Spandolf, C., Weissenborn, M. J., , Grogan, G. & Hauer, B. Structure-Guided Redesign of CYP153AM.aq for the Improved Terminal Hydroxylation of Fatty Acids.  ChemCatChem 8, 3234-3239, (2016) DOI: 10.1002/cctc.201600680

The structure of a P450 ω-hydroxylase bound to its fatty acid product was determined, which revealed a narrow substrate tunnel that leads to the heme. The introduction of an arginine side chain in proximity to the carboxyl group of the fatty acid led to a reduced KM value for dodecanoic acid, which suggests the importance of an anchoring point in the active site. An increase in the flexibility of the substrate recognition region was also engineered, which resulted in a threefold improved product formation.

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