An artificial heme enzyme was created through self‐assembly from hemin and the lactococcal multidrug resistance regulator (LmrR). The crystal structure shows the heme bound inside the hydrophobic ...pore of the protein, where it appears inaccessible for substrates. However, good catalytic activity and moderate enantioselectivity was observed in an abiological cyclopropanation reaction. We propose that the dynamic nature of the structure of the LmrR protein is key to the observed activity. This was supported by molecular dynamics simulations, which showed transient formation of opened conformations that allow the binding of substrates and the formation of pre‐catalytic structures.
Get a move on: An artificial heme enzyme based on the lactococcal multidrug resistance regulator (LmrR; blue/green) was created through self‐assembly and shows good activity in cyclopropanation reactions. The dynamics of the system are of key importance for its activity.
The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding ...pocket. Such fine‐tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (kcat) of the designer enzyme by almost 100‐fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200‐fold.
Designer enzyme: The directed evolution of a designer enzyme featuring a uniquely reactive aniline side chain as catalytic residue (in red) is reported. Multiple beneficial mutations were identified (blue), which when combined increase the turnover frequency (kcat) of the designer enzyme by more than 90 times.
Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric ...4‐oxalocrotonate tautomerase (4‐OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem‐fused 4‐OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4‐OT was then subjected to eleven rounds of directed evolution to give variant 4‐OT(F11), which showed an up to 320‐fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4‐OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180° relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins.
Less symmetry, more diversity: Here we show that gene duplication and fusion to break structural symmetry, allowing independent sequence diversification of neighboring subunits by directed evolution, is a powerful strategy to boost catalysis by a promiscuous homohexameric 4‐oxalocrotonate tautomerase (4‐OT), enabling efficient asymmetric Michael additions.
Cytochrome P450 monooxygenases (P450s) are attractive enzymes for the pharmaceutical industry, in particular, for applications in steroidal drug synthesis. Here, we report a comprehensive functional ...and structural characterization of CYP109E1, a novel steroid‐converting cytochrome P450 enzyme identified from the genome of Bacillus megaterium DSM319. In vitro and whole‐cell in vivo turnover experiments, combined with binding assays, revealed that CYP109E1 is able to hydroxylate testosterone at position 16β. Related steroids with bulky substituents at carbon C17, like corticosterone, bind to the enzyme without being converted. High‐resolution X‐ray structures were solved of a steroid‐free form of CYP109E1 and of complexes with testosterone and corticosterone. The structural analysis revealed a highly dynamic active site at the distal side of the heme, which is wide open in the absence of steroids, can bind four ordered corticosterone molecules simultaneously, and undergoes substantial narrowing upon binding of single steroid molecules. In the crystal structures, the single bound steroids adopt unproductive binding modes coordinating the heme‐iron with their C3‐keto oxygen. Molecular dynamics (MD) simulations suggest that the steroids may also bind in ~180° reversed orientations with the C16 carbon and C17‐substituents pointing toward the heme, leading to productive binding of testosterone explaining the observed regio‐ and stereoselectivity. The X‐ray structures and MD simulations further identify several residues with important roles in steroid binding and conversion, which could be confirmed by site‐directed mutagenesis. Taken together, our results provide unique insights into the CYP109E1 activity, substrate specificity, and regio/stereoselectivity.
Database
The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5L90 (steroid‐free CYP109E1), 5L91 (CYP109E1‐COR4), 5L94 (CYP109E1‐TES), and 5L92 (CYP109E1‐COR).
Enzymes
Cytochrome P450 monooxygenase CYP109E1, EC 1.14.14.1, UniProt ID: D5DKI8, Adrenodoxin reductase EC 1.18.1.6.
Comprehensive functional and structural characterization of the steroid‐specific P450 monooxygenase CYP109E1. The bacterial enzyme converts testosterone to 16β‐hydroxytestosterone with high regio‐ and stereoselectivity. Open and closed crystal structures were determined, related to substrate‐free and steroid‐bound states. Our data, combined with results from MD simulations and site‐directed mutagenesis, provide insights explaining CYP109E1 activity, substrate specificity, and regio/stereoselectivity.
Aspartic acid derivatives with branched N‐alkyl or N‐arylalkyl substituents are valuable precursors to artificial dipeptide sweeteners such as neotame and advantame. The development of a biocatalyst ...to synthesize these compounds in a single asymmetric step is an as yet unmet challenge. Reported here is an enantioselective biocatalytic synthesis of various difficult N‐substituted aspartic acids, including N‐(3,3‐dimethylbutyl)‐l‐aspartic acid and N‐3‐(3‐hydroxy‐4‐methoxyphenyl)propyl‐l‐aspartic acid, precursors to neotame and advantame, respectively, using an engineered variant of ethylenediamine‐N,N′‐disuccinic acid (EDDS) lyase from Chelativorans sp. BNC1. This engineered C–N lyase (mutant D290M/Y320M) displayed a remarkable 1140‐fold increase in activity for the selective hydroamination of fumarate compared to that of the wild‐type enzyme. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step‐economic synthesis of an important class of food additives.
Sweet! The enzyme ethylenediamine‐N,N′‐disuccinic acid lyase was optimized by structure‐guided mutagenesis for the enantioselective synthesis of challenging N‐substituted aspartic acids, which are important chiral precursors to artificial dipeptide sweeteners such as neotame and advantame. This redesigned C–N lyase displayed a remarkable 1140‐fold increase in activity for selective hydroamination of fumarate.
Photosystem II (PSII) is a light-driven protein, involved in the primary reactions of photosynthesis. In plant photosynthetic membranes PSII forms large multisubunit supercomplexes, containing a ...dimeric core and up to four light-harvesting complexes (LHCs), which act as antenna proteins. Here we solved a three-dimensional (3D) structure of the C
S
M
supercomplex from Arabidopsis thaliana using cryo-transmission electron microscopy (cryo-EM) and single-particle analysis at an overall resolution of 5.3 Å. Using a combination of homology modelling and restrained refinement against the cryo-EM map, it was possible to model atomic structures for all antenna complexes and almost all core subunits. We located all 35 chlorophylls of the core region based on the cyanobacterial PSII structure, whose positioning is highly conserved, as well as all the chlorophylls of the LHCII S and M trimers. A total of 13 and 9 chlorophylls were identified in CP26 and CP24, respectively. Energy flow from LHC complexes to the PSII reaction centre is proposed to follow preferential pathways: CP26 and CP29 directly transfer to the core using several routes for efficient transfer; the S trimer is directly connected to CP43 and the M trimer can efficiently transfer energy to the core through CP29 and the S trimer.
The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 (VD3) to 25-hydroxyvitamin D3. Here, we show that this enzyme is also able to convert ...testosterone in a highly regio- and stereoselective manner to 16β-hydroxytestosterone. To reveal the structural determinants governing the regio- and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a nonproductive site away from the heme-iron. To examine whether the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics (MD) simulations were performed with testosterone and vitamin D3 (VD3) present in the active site. These MD simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon-hydrogen bond was taken into account. Overall, the first-time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site-directed mutagenesis toward improved enzymatic properties of this enzyme.
Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as ...well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability‐landscape approach that identified three single mutations (R11Y, R11I and A33D) within the enzyme 4‐oxalocrotonate tautomerase (4‐OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature Tm (up to 20 °C) and in half‐life at 80 °C (up to 111‐fold). Introduction of these beneficial mutations in an enantioselective but thermolabile 4‐OT variant (M45Y/F50A) afforded improved triple‐mutant enzyme variants showing an up to 39 °C increase in Tm value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability‐landscape‐guided protein engineering for thermostabilizing enzymes.
Hotspot for enzymes! The enzyme 4‐oxalocrotonate tautomerase (4‐OT) promiscuously catalyzes synthetically useful Michael‐type addition reactions. A mutability‐landscape approach was applied to identify “hotspot” positions within 4‐OT at which single mutations resulted in enzyme variants with strongly improved thermostability. This information was used to engineer a 4‐OT variant with greatly increased apparent Tm but no reduction in activity or enantioselectivity.
Transaminases are attractive catalysts for the production of enantiopure amines. However, the poor stability of these enzymes often limits their application in biocatalysis. Here, we used a framework ...for enzyme stability engineering by computational library design (FRESCO) to stabilize the homodimeric PLP fold type I ω-transaminase from Pseudomonas jessenii. A large number of surface-located point mutations and mutations predicted to stabilize the subunit interface were examined. Experimental screening revealed that 10 surface mutations out of 172 tested were indeed stabilizing (6% success), whereas testing 34 interface mutations gave 19 hits (56% success). Both the extent of stabilization and the spatial distribution of stabilizing mutations showed that the subunit interface was critical for stability. After mutations were combined, 2 very stable variants with 4 and 6 mutations were obtained, which in comparison to wild type (T m app = 62 °C) displayed T m app values of 80 and 85 °C, respectively. These two variants were also 5-fold more active at their optimum temperatures and tolerated high concentrations of isopropylamine and cosolvents. This allowed conversion of 100 mM acetophenone to (S)-1-phenylethylamine (>99% enantiomeric excess) with high yield (92%, in comparison to 24% with the wild-type transaminase). Crystal structures mostly confirmed the expected structural changes and revealed that the most stabilizing mutation, I154V, featured a rarely described stabilization mechanism: namely, removal of steric strain. The results show that computational interface redesign can be a rapid and powerful strategy for transaminase stabilization.
In proteins, the amino acids Phe, Tyr, and especially Trp are frequently involved in π interactions such as π–π, cation−π, and CH−π bonds. These interactions are often crucial for protein structure ...and protein–ligand binding. A powerful means to study these interactions is progressive fluorination of these aromatic residues to modulate the electrostatic component of the interaction. However, to date no protein expression platform is available to produce milligram amounts of proteins labeled with such fluorinated amino acids. Here, we present a Lactococcus lactis Trp auxotroph-based expression system for efficient incorporation (≥95%) of mono-, di-, tri-, and tetrafluorinated, as well as a methylated Trp analog. As a model protein we have chosen LmrR, a dimeric multidrug transcriptional repressor protein from L. lactis. LmrR binds aromatic drugs, like daunomycin and riboflavin, between Trp96 and Trp96′ in the dimer interface. Progressive fluorination of Trp96 decreased the affinity for the drugs 6- to 70-fold, clearly establishing the importance of electrostatic π–π interactions for drug binding. Presteady state kinetic data of the LmrR–drug interaction support the enthalpic nature of the interaction, while high resolution crystal structures of the labeled protein–drug complexes provide for the first time a structural view of the progressive fluorination approach. The L. lactis expression system was also used to study the role of Trp68 in the binding of riboflavin by the membrane-bound riboflavin transport protein RibU from L. lactis. Progressive fluorination of Trp68 revealed a strong electrostatic component that contributed 15–20% to the total riboflavin-RibU binding energy.
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