Benjamin R. Lichman

Identification of adult nephron progenitors capable of kidney regeneration in zebrafish

Loss of kidney function underlies many renal diseases. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10–30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies.

One-pot triangular chemoenzymatic cascades for the syntheses of chiral alkaloids from dopamine

We describe novel chemoenzymatic routes to (S)-benzylisoquinoline and (S)-tetrahydroprotoberberine alkaloids using the enzymes transaminase (TAm) and norcoclaurine synthase (NCS) in a one-pot, one-substrate ‘triangular’ cascade. Employment of up to two C–C bond forming steps allows for the rapid generation of molecular complexity under mild conditions.

‘Dopamine-first’ mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile

Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the Pictet–Spengler condensation of dopamine and an aldehyde, forming a substituted (S)‐tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both in vitro biocatalysis and in vivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two proposed mechanisms for NCS activity: (a) one based on the holo X‐ray crystal structure and (b) the ‘dopamine‐first’ mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine‐first mechanism. Suppression of the non‐enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzymes aldehyde activity profile. This both verifies the dopamine‐first mechanism and demonstrates the potential for the rational engineering of NCS activity.

Enzymatic and Chemoenzymatic Three-Step Cascades for the Synthesis of Stereochemically Complementary Trisubstituted Tetrahydroisoquinolines

Abstract Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4‐trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step‐efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3‐hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet–Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)‐metaraminol. Subsequent cyclization catalyzed either enzymatically by a norcoclaurine synthase or chemically by phosphate resulted in opposite stereoselectivities in the products at the C1 position, thus providing access to both orientations of the THIQ C1 substituent. This highlights the importance of selecting from both chemo‐ and biocatalysts for optimal results.

Enzyme catalysed Pictet-Spengler formation of chiral 1,1’-disubstituted- and spiro-tetrahydroisoquinolines

The Pictet–Spengler reaction (PSR) involves the condensation and ring closure between a β-arylethylamine and a carbonyl compound. The combination of dopamine and ketones in a PSR leads to the formation of 1,1′-disubstituted tetrahydroisoquinolines (THIQs), structures that are challenging to synthesize and yet are present in a number of bioactive natural products and synthetic pharmaceuticals. Here we have discovered that norcoclaurine synthase from Thalictrum flavum (TfNCS) can catalyse the PSR between dopamine and unactivated ketones, thus facilitating the facile biocatalytic generation of 1,1′-disubstituted THIQs. Variants of TfNCS showing improved conversions have been identified and used to synthesize novel chiral 1,1′-disubstituted and spiro-THIQs. Enzyme catalysed PSRs with unactivated ketones are unprecedented, and, furthermore, there are no equivalent stereoselective chemical methods for these transformations. This discovery advances the utility of enzymes for the generation of diverse THIQs in vitro and in vivo.

The folding of a family of three-helix bundle proteins: spectrin R15 has a robust folding nucleus, unlike its homologous neighbours.

Three homologous spectrin domains have remarkably different folding characteristics. We have previously shown that the slow-folding R16 and R17 spectrin domains can be altered to resemble the fast folding R15, in terms of speed of folding (and unfolding), landscape roughness and folding mechanism, simply by substituting five residues in the core. Here we show that, by contrast, R15 cannot be engineered to resemble R16 and R17. It is possible to engineer a slow-folding version of R15, but our analysis shows that this protein neither has a rougher energy landscape nor does change its folding mechanism. Quite remarkably, R15 appears to be a rare example of a protein with a folding nucleus that does not change in position or in size when its folding nucleus is disrupted. Thus, while two members of this protein family are remarkably plastic, the third has apparently a restricted folding landscape.

Structural evidence for the dopamine-first mechanism of norcoclaurine synthase

Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocatalyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism-inspired ligand. The structure supports two key features of the NCS “dopamine-first” mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a previously unobserved ligand-bound conformation that may be catalytically significant. The potential roles of inhibitory binding and alternative amino acid conformations in the mechanism have also been revealed. This work significantly advances our understanding of the NCS mechanism and will aid future efforts to engineer the substrate scope and catalytic properties of this useful biocatalyst.

Identification of iridoid synthases from Nepeta species: Iridoid cyclization does not determine nepetalactone stereochemistry

Nepetalactones are iridoid monoterpenes with a broad range of biological activities produced by plants in the Nepeta genus. However, none of the genes for nepetalactone biosynthesis have been discovered. Here we report the transcriptomes of two Nepeta species, each with distinctive profiles of nepetalactone stereoisomers. As a starting point for investigation of nepetalactone biosynthesis in Nepeta, these transcriptomes were used to identify candidate genes for iridoid synthase homologs, an enzyme that has been shown to form the core iridoid skeleton in several iridoid producing plant species. Iridoid synthase homologs identified from the transcriptomes were cloned, heterologously expressed, and then assayed with the 8-oxogeranial substrate. These experiments revealed that catalytically active iridoid synthase enzymes are present in Nepeta, though there are unusual mutations in key active site residues. Nevertheless, these enzymes exhibit similar catalytic activity and product profile compared to previously reported iridoid synthases from other plants. Notably, four nepetalactone stereoisomers with differing stereochemistry at the 4α and 7α positions – which are generated during the iridoid synthase reaction – are observed at different ratios in various Nepeta species. This work strongly suggests that the variable stereochemistry at these 4α and 7α positions of nepetalactone diastereomers is established further downstream in the iridoid pathway in Nepeta. Overall, this work provides a gateway into the biosynthesis of nepetalactones in Nepeta.

Point of View: A transatlantic perspective on 20 emerging issues in biological engineering

Advances in biological engineering are likely to have substantial impacts on global society. To explore these potential impacts we ran a horizon scanning exercise to capture a range of perspectives on the opportunities and risks presented by biological engineering. We first identified 70 potential issues, and then used an iterative process to prioritise 20 issues that we considered to be emerging, to have potential global impact, and to be relatively unknown outside the field of biological engineering. The issues identified may be of interest to researchers, businesses and policy makers in sectors such as health, energy, agriculture and the environment.

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyses the activation of its substrate 8-oxogeranial into a reactive enol intermediate but does not catalyse the subsequent cyclisation into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) which catalyse the stereoselective cyclisation of the enol intermediate into nepetalactol isomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effect in cats. Structural characterisation of the NEPS3 cyclase reveals it binds to NAD+ yet does not utilise it chemically for a non-oxidoreductive formal [4+2] cyclisation. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.