Late Microaerobic Growth for Efficient Production of Human Cytochrome P450 3A4 in E. coli.

Detailed preclinical characterization of metabolites formed in vivo from candidate drug substances is mandatory prior to the initiation of clinical trials. Therefore, inexpensive and efficient methods for drug metabolite synthesis are of high importance for rapid advancement of the drug development process. A large fraction of small molecule drugs is modified by monooxygenase cytochrome P450 3A4 produced in the human liver and intestine. Therefore, this enzyme is frequently employed to catalyze metabolite synthesis in vitro, making 3A4 availability a critical requirement in early drug development. Unfortunately, the recombinant production of this enzyme in microbial hosts is notoriously difficult. Maintaining low oxygen transfer rates and the use of rich media for host cultivation are required for P450 3A4 production. However, detailed studies on the relationship between oxygen supply and P450 3A4 space-time yields are missing. We describe an improved biotechnological process for the heterologous expression of P450 3A4 together with its redox partner, cytochrome P450 reductase, in Escherichia coli. Enzyme production was most efficient under so-called "late microaerobic" growth conditions, in which the cells have just not yet made the switch to anaerobic metabolism, characterized by a limited oxygen supply leading to oxygen concentrations in the liquid phase that are far below the detection limit of standard oxygen electrodes. Furthermore, feeding the carbon source glycerol as well as controlling cellular acetate formation improved process productivity. The presented protocol resulted in the formation of functional recombinant 3A4 at concentrations up to 680 nmol L-1.

Carbene-Induced Rescue of Catalytic Activity in Deactivated Nitrite Reductase Mutant.

The role of His145 in the T1 copper center of nitrite reductase (NiR) is pivotal for the activity of the enzyme. Mutation to a glycine at this position enables the reconstitution of the T1 center by the addition of imidazole as exogenous ligands, however the catalytic activity is only marginally rescued. Here, we demonstrate that the uptake of 1,3-dimethylimidazolylidene as N-heterocyclic carbene (NHC) by the H145G NiR mutant instead of imidazole yields a significantly more active catalyst, suggesting a beneficial role of such C-bonding. Spectroscopic analyses of the formed H145G≈NHC variant as well as an analogue without the catalytic T2 copper center reveal no significant alteration of the T1 site compared to the wild type or the variant containing imidazole as exogenous N-bound surrogate of H145. However, the presence of the carbene doubles the catalytic activity of the mutant compared to the imidazole variant. This enhanced activity has been attributed to a faster electron transfer to the T1 center in the NHC variant and a concomitant change of the rate-limiting step.

Carbene in Cupredoxin Protein Scaffolds: Replacement of a Histidine Ligand in the Active Site Substantially Alters Copper Redox Properties.

N-heterocyclic carbene (NHC) ligands have had a major impact in homogeneous catalysis, however, their potential role in biological systems is essentially unexplored. We replaced a copper-coordinating histidine (His) in the active site of the redox enzyme azurin with exogenous dimethyl imidazolylidene. This NHC rapidly restores the type-1 Cu center, with spectroscopic properties (EPR, UV/Vis) that are identical to those from N-coordination of the His in the wild type. However, the introduction of the NHC markedly alters the redox potential of the metal, which is a key functionality of this blue copper protein. These results suggest that C-bonding for histidine is plausible and a potentially relevant bonding mode of redox-active metalloenzymes in their (transient) active states.

Stereoelectronic effects in the reaction of aromatic substrates catalysed by Halomonas elongata transaminase and its mutants.

A transaminase from Halomonas elongata and four mutants generated by an in silico-based design were recombinantly produced in E. coli, purified and applied to the amination of mono-substituted aromatic carbonyl-derivatives. While benzaldehyde derivatives were excellent substrates, only NO2-acetophenones were transformed into the (S)-amine with a high enantioselectivity. The different behaviour of wild-type and mutated transaminases was assessed by in silico substrate binding mode studies.

Minimalistic peptidic scaffolds harbouring an artificial carbene-containing amino acid modulate reductase activity.

Inspired by the boom of new artificial metalloenzymes, we developed an Fmoc-protected histidinium salt (Hum) as N-heterocyclic carbene precursor. Hum was placed via solid-phase peptide synthesis into short 7-mer peptides. Upon iridation, the metallo-peptidic construct displayed activity in catalytic hydrogenation that outperforms small molecule analogues and which is dependent on the peptide sequence, a typical feature of metalloenzymes.

An alginate-confined peroxygenase-CLEA for styrene epoxidation.

Oxyfunctionalisation reactions in neat substrate still pose a challenge for biocatalysis. Here, we report an alginate-confined peroxygenase-CLEA to catalyse the enantioselective epoxidation of cis-ß-methylstyrene in a solvent-free reaction system achieving turnover numbers of 96 000 for the biocatalyst and epoxide concentrations of 48 mM.

Widely applicable background depletion step enables transaminase evolution through solid-phase screening.

Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications. This process, however, is challenged by the limited availability of effective high-throughput protocols to evaluate mutant libraries. Here we report a rapid, reliable, and widely applicable background depletion method for solid-phase screening of transaminase variants, which was successfully applied to a transaminase from Halomonas elongata (HEWT), evolved through rounds of random mutagenesis towards a series of diverse prochiral ketones. This approach enabled the identification of transaminase variants in viable cells with significantly improved activity towards para-substituted acetophenones (up to 60-fold), as well as tetrahydrothiophen-3-one and related substrates. Rationalisation of the mutants was assisted by determination of the high-resolution wild-type HEWT crystal structure presented herein.

Strategic single point mutation yields a solvent- and salt-stable transaminase from Virgibacillus sp. in soluble form.

A new transaminase (VbTA) was identified from the genome of the halotolerant marine bacterium Virgibacillus 21D. Following heterologous expression in Escherichia coli, it was located entirely in the insoluble fraction. After a single mutation, identified via sequence homology analyses, the VbTA T16F mutant was successfully expressed in soluble form and characterised. VbTA T16F showed high stability towards polar organic solvents and salt exposure, accepting mainly hydrophobic aromatic amine and carbonyl substrates. The 2.0 Å resolution crystal structure of VbTA T16F is here reported, and together with computational calculations, revealed that this mutation is crucial for correct dimerisation and thus correct folding, leading to soluble protein expression.

Synthesis and in-vitro anticancer evaluation of polyarsenicals related to the marine sponge derived Arsenicin A.

In the light of the promising bioactivity of the tetraarsenic marine metabolite arsenicin A, the dimethyl analogue 2 and four isomeric methylene homologues (including the natural product itself) were obtained using a one-pot microwave-assisted synthesis, starting from arsenic (III) oxide. Due to the poor diagnostic value of the NMR technique in the structural elucidation of these molecules, they were fully characterized by mass spectrometry and infrared (IR)-spectroscopy, comparing density functional theory (DFT) simulated and experimental spectra. This synthetic procedure provided a fast and efficient access to the cytotoxicity evaluation of organoarsenical leads of the natural hit molecule. From in vitro screening, each tested compound resulted in being more active than the FDA-approved arsenic trioxide, with the most lipophilic molecule in the series showing the best growth inhibition of both leukemia and solid tumor cell lines. These results may open promising perspectives in the development of new more potent and selective arsenical drugs against solid tumors.