Efficient synthesis of enantiopure amines from alcohols using resting E. coli cells and ammonia.

α-Chiral amines are pivotal building blocks for chemical manufacturing. Stereoselective amination of alcohols is receiving increased interest due to its higher atom-efficiency and overall improved environmental footprint compared with other chemocatalytic and biocatalytic methods. We previously developed a hydrogen-borrowing amination by combining an alcohol dehydrogenase (ADH) with an amine dehydrogenase (AmDH) in vitro. Herein, we implemented the ADH-AmDH bioamination in resting Escherichia coli cells for the first time. Different genetic constructs were created and tested in order to obtain balanced expression levels of the dehydrogenase enzymes in E. coli. Using the optimized constructs, the influence of several parameters towards the productivity of the system were investigated such as the intracellular NAD+/NADH redox balance, the cell loading, the survival rate of recombinant E. coli cells, the possible toxicity of the components of the reaction at different concentrations and the influence of different substrates and cosolvents. In particular, the cofactor redox-balance for the bioamination was maintained by the addition of moderate and precise amounts of glucose. Higher concentrations of certain amine products resulted in toxicity and cell death, which could be alleviated by the addition of a co-solvent. Notably, amine formation was consistent using several independently grown E. coli batches. The optimized E. coli/ADH-AmDH strains produced enantiopure amines from the alcohols with up to 80% conversion and a molar productivity up to 15 mM. Practical applicability was demonstrated in a gram-scale biotransformation. In summary, the present E. coli-ADH-AmDH system represents an important advancement towards the development of 'green', efficient and selective biocatalytic processes for the amination of alcohols.

Lysosomes nor Mice Move Forward without Borcs7.

Lysosome function and position in the cytoplasm depends on the BORCS machinery, which tethers lysosomes to the kinesin microtubule motor. A recent paper of Snouwaert et al. in Cell Reports characterizes a mouse with a spontaneous mutation in the Borcs7 subunit, which causes axonal dystrophy and impaired motor function.

Concurrent presence of on- and off-pathway folding intermediates of apoflavodoxin at physiological ionic strength.

Flavodoxins have a protein topology that can be traced back to the universal ancestor of the three kingdoms of life. Proteins with this type of architecture tend to temporarily misfold during unassisted folding to their native state and form intermediates. Several of these intermediate species are molten globules (MGs), which are characterized by a substantial amount of secondary structure, yet without the tertiary side-chain packing of natively folded proteins. An off-pathway MG is formed at physiological ionic strength in the case of the F44Y variant of Azotobacter vinelandii apoflavodoxin (i.e., flavodoxin without flavin mononucleotide (FMN)). Here, we show that at this condition actually two folding species of this apoprotein co-exist at equilibrium. These species were detected by using a combination of FMN fluorescence quenching upon cofactor binding to the apoprotein and of polarized time-resolved tryptophan fluorescence spectroscopy. Besides the off-pathway MG, we observe the simultaneous presence of an on-pathway folding intermediate, which is native-like. Presence of concurrent intermediates at physiological ionic strength enables future exploration of how aspects of the cellular environment, like for example involvement of chaperones, affect these species.

Folding of proteins with a flavodoxin-like architecture.

The flavodoxin-like fold is a protein architecture that can be traced back to the universal ancestor of the three kingdoms of life. Many proteins share this α-ß parallel topology and hence it is highly relevant to illuminate how they fold. Here, we review experiments and simulations concerning the folding of flavodoxins and CheY-like proteins, which share the flavodoxin-like fold. These polypeptides tend to temporarily misfold during unassisted folding to their functionally active forms. This susceptibility to frustration is caused by the more rapid formation of an α-helix compared to a ß-sheet, particularly when a parallel ß-sheet is involved. As a result, flavodoxin-like proteins form intermediates that are off-pathway to native protein and several of these species are molten globules (MGs). Experiments suggest that the off-pathway species are of helical nature and that flavodoxin-like proteins have a nonconserved transition state that determines the rate of productive folding. Folding of flavodoxin from Azotobacter vinelandii has been investigated extensively, enabling a schematic construction of its folding energy landscape. It is the only flavodoxin-like protein of which cotranslational folding has been probed. New insights that emphasize differences between in vivo and in vitro folding energy landscapes are emerging: the ribosome modulates MG formation in nascent apoflavodoxin and forces this polypeptide toward the native state.

The Ribosome Restrains Molten Globule Formation in Stalled Nascent Flavodoxin.

Folding of proteins usually involves intermediates, of which an important type is the molten globule (MG). MGs are ensembles of interconverting conformers that contain (non-)native secondary structure and lack the tightly packed tertiary structure of natively folded globular proteins. Whereas MGs of various purified proteins have been probed to date, no data are available on their presence and/or effect during protein synthesis. To study whether MGs arise during translation, we use ribosome-nascent chain (RNC) complexes of the electron transfer protein flavodoxin. Full-length isolated flavodoxin, which contains a non-covalently bound flavin mononucleotide (FMN) as cofactor, acquires its native α/ß parallel topology via a folding mechanism that contains an off-pathway intermediate with molten globular characteristics. Extensive population of this MG state occurs at physiological ionic strength for apoflavodoxin variant F44Y, in which a phenylalanine at position 44 is changed to a tyrosine. Here, we show for the first time that ascertaining the binding rate of FMN as a function of ionic strength can be used as a tool to determine the presence of the off-pathway MG on the ribosome. Application of this methodology to F44Y apoflavodoxin RNCs shows that at physiological ionic strength the ribosome influences formation of the off-pathway MG and forces the nascent chain toward the native state.

Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome.

Correct folding of proteins is crucial for cellular homeostasis. More than thirty percent of proteins contain one or more cofactors, but the impact of these cofactors on co-translational folding remains largely unknown. Here, we address the binding of flavin mononucleotide (FMN) to nascent flavodoxin, by generating ribosome-arrested nascent chains that expose either the entire protein or C-terminally truncated segments thereof. The native α/ß parallel fold of flavodoxin is among the most ancestral and widely distributed folds in nature and exploring its co-translational folding is thus highly relevant. In Escherichia coli (strain BL21(DE3) Δtig::kan) FMN turns out to be limiting for saturation of this flavoprotein on time-scales vastly exceeding those of flavodoxin synthesis. Because the ribosome affects protein folding, apoflavodoxin cannot bind FMN during its translation. As a result, binding of cofactor to released protein is the last step in production of this flavoprotein in the cell. We show that once apoflavodoxin is entirely synthesized and exposed outside the ribosome to which it is stalled by an artificial linker containing the SecM sequence, the protein is natively folded and capable of binding FMN.

Impact of residues remote from the catalytic centre on enzyme catalysis of copper nitrite reductase.

Enzyme mechanisms are often probed by structure-informed point mutations and measurement of their effects on enzymatic properties to test mechanistic hypotheses. In many cases, the challenge is to report on complex, often inter-linked elements of catalysis. Evidence for long-range effects on enzyme mechanism resulting from mutations remains sparse, limiting the design/redesign of synthetic catalysts in a predictable way. Here we show that improving the accessibility of the active site pocket of copper nitrite reductase by mutation of a surface-exposed phenylalanine residue (Phe306), located 12 Å away from the catalytic site type-2 Cu (T2Cu), profoundly affects intra-molecular electron transfer, substrate-binding and catalytic activity. Structures and kinetic studies provide an explanation for the lower affinity for the substrate and the alteration of the rate-limiting step in the reaction. Our results demonstrate that distant residues remote from the active site can have marked effects on enzyme catalysis, by driving mechanistic change through relatively minor structural perturbations.