Solution structures and effects of a platinum compound successively bound MYC G-quadruplex.

G-quadruplex (G4) structures play integral roles in modulating biological functions and can be regulated by small molecules. The MYC gene is critical during tumor initiation and malignant progression, in which G4 acts as an important modulation motif. Herein, we reported the MYC promoter G4 recognized by a platinum(II) compound Pt-phen. Two Pt-phen-MYC G4 complex structures in 5 mM K+ were determined by NMR. The Pt-phen first strongly binds the 3'-end of MYC G4 to form a 1:1 3'-end binding complex and then binds 5'-end to form a 2:1 complex with more Pt-phen. In the complexes, the Pt-phen molecules are well-defined and stack over four bases at the G-tetrad for a highly extensive π-π interaction, with the Pt atom aligning with the center of the G-tetrad. The flanking residues were observed to rearrange and cover on top of Pt-phen to stabilize the whole complex. We further demonstrated that Pt-phen targets G4 DNA in living cells and represses MYC gene expression in cancer cells. Our work elucidated the structural basis of ligand binding to MYC promoter G4. The platinum compound bound G4 includes multiple complexes formation, providing insights into the design of metal ligands targeting oncogene G4 DNA.

Best method to determine DNA G-quadruplex folding: The 1H-13C HSQC NMR experiment.

NMR spectroscopy is the major method for G-quadruplex structure determination under physiologically relevant solution conditions. Unlike duplex B-DNA, in which all nucleotides adopt an anti glycosidic conformation, the core tetrad-guanines in a G-quadruplex can adopt anti or syn glycosidic conformation depending on the folding structure. An experimental method that can clearly and unambiguously determine syn and anti tetrad-Gs in a G-quadruplex is highly desirable and necessary. In the present study, we exploit the advantages of the 1H-13C HSQC experiment to determine tetrad-G's glycosidic conformation and thus folding topology of G-quadruplexes. We use several examples to demonstrate the clear and straightforward determination of the guanine glycosidic conformations and G-quadruplex folding structures. Moreover, 1H-13C HSQC data can readily identify adenine H2 resonances as well as determine unusual syn conformation in loop and flanking sequences, a challenging task by standard 2D NOESY.

Gas-Phase Fragmentation as a Probe of G-Quadruplex Formation.

G-quadruplex (G4) DNA is found in oncogene promoters and human telomeres and is an attractive anticancer target. Stable G4 structures form in guanine-rich sequences in the presence of metal cations and can stabilize further with specific ligand adduction. To explore the preservation and stability of this secondary structure with mass spectrometry, gas-phase collision-induced dissociation kinetics of G4-like and non-G4-like ion structures were determined in a linear quadrupole ion trap. This study focused on a sequence from the promoter of the MYC oncogene, MycG4, and a mutant non-G4-forming sequence, MycNonG4. At relatively high ion activation energies, the backbone fragmentation patterns of the MycG4 and MycNonG4 are similar, while potassium ion-stabilized G4-folded [MycG4 + 2K-7H]5- and counterpart [MycG4-5H]5- ions are essentially indistinguishable, indicating that high-energy fragmentation is not sensitive to the G4 structure. At low energies, the backbone fragmentation patterns of MycG4 and MycNonG4 are significantly different. For MycG4, fragmentation over time differed significantly between the potassium-bound and free structures, reflecting the preservation of the G4 structure in the gas phase. Kinetic measurements revealed the [MycG4 + 2K-7H]5- ions to fragment two to three times more slowly than the [MycG4-5H]5-. Results for the control MycNonG4 indicated that the phenomena noted for [MycG4 + 2K-7H]5- ions are specific to G4-folding. Therefore, our data show that gentle activation conditions can lead to fragmentation behavior that is sensitive to G-quadruplex structure, revealing differences in kinetic stabilities of isomeric structures as well as the regions of the sequence that are directly involved in forming these structures.

DNA G-Quadruplex in Human Telomeres and Oncogene Promoters: Structures, Functions, and Small Molecule Targeting.

DNA G-quadruplex secondary structures formed in guanine-rich human telomeres and oncogene promoters are functionally important and have emerged as a promising new class of cancer-specific drug targets. These globular intramolecular structures are stabilized by K+ or Na+ and form readily under physiological solution conditions. Moreover, G-quadruplexes are epigenetic features and can alter chromatin structure and function together with interactive proteins. Here, we discuss our efforts over the last two decades to understand the structures and functions of DNA G-quadruplexes formed in key oncogene promoters and human telomeres and their interactions with small molecules. Using high-field NMR spectroscopy, we determined the high-resolution structures of physiologically relevant telomeric G-quadruplexes in K+ solution with a major form (hybrid-2) and a minor form (hybrid-1), as well as a two-tetrad intermediate. The intrinsic structural polymorphism of telomeric DNA may be important for the biology of human telomeres, and we proposed a model for the interconversion. More recently, we have worked on G-quadruplexes of MYC, BCL2, PDGFR-ß, VEGF, and k-RAS oncogene promoters. We determined the structure of the major G-quadruplex formed in the MYC promoter, a prototype for parallel G-quadruplexes. It is the first example of the parallel-stranded G3NG3 structure motif with a 1-nt loop, which is prevalent in promoter sequences and likely evolutionarily selected to initiate folding. Remarkably, the parallel MYC promoter G-quadruplexes are highly stable. Additionally, we determined the molecular structures of G-quadruplexes formed in human BCL2, VEGF, and PDGFR-ß promoters, each adopting a unique structure. For example, the BCL2 promoter contains distinct interchangeable G-quadruplexes in two adjacent regions, suggesting precise regulation by different proteins. The PDGFR-ß promoter adopts unique "broken-strand" and vacancy G-quadruplexes, which can be recognized by cellular guanine metabolites for a potential regulatory role.Structural information on G-quadruplexes in complex with small-molecules is critical for understanding specific recognition and structure-based rational drug design. Our studies show that many G-quadruplexes contain unique structural features such as capping and loop structures, allowing specific recognition by drugs and protein. This represents a paradigm shift in understanding DNA as a drug target: Rather than a uniform, nonselective binding site in duplex DNA, the G-quadruplex is being pursued as a new class of selectively targetable drug receptors. We focus on targeting the biologically relevant MYC promoter G-quadruplex (MycG4) with small molecules and have determined its first and additional drug complex structures. Very recently, we have discovered clinically tested indenoisoquinolines as strong MycG4 binders and potent MYC inhibitors. We have also discovered drugs targeting the unique dGMP-bound-vG4 formed in the PDGFR-ß promoter. Moreover, we determined the complex structures of the first small molecules that specifically recognize the physiologically relevant human telomeric G-quadruplexes. Unlike the previously recognized dogma that the optimal G-quadruplex ligands are large aromatic or cyclic compounds, our results suggest that smaller asymmetric compounds with appropriate functional groups are better choices to specifically bind G-quadruplexes. This body of work lays a strong foundation for future work aimed at understanding the cellular functions of G-quadruplexes and G-quadruplex-targeted drug design.

Structural recognition of the MYC promoter G-quadruplex by a quinoline derivative: insights into molecular targeting of parallel G-quadruplexes.

DNA G-Quadruplexes (G4s) formed in oncogene promoters regulate transcription. The oncogene MYC promoter G4 (MycG4) is the most prevalent G4 in human cancers. However, the most studied MycG4 sequence bears a mutated 3'-residue crucial for ligand recognition. Here, we report a new drug-like small molecule PEQ without a large aromatic moiety that specifically binds MycG4. We determined the NMR solution structures of the wild-type MycG4 and its 2:1 PEQ complex, as well as the structure of the 2:1 PEQ complex of the widely used mutant MycG4. Comparison of the two complex structures demonstrates specific molecular recognition of MycG4 and shows the clear effect of the critical 3'-mutation on the drug binding interface. We performed a systematic analysis of the four available complex structures involving the same mutant MycG4, which can be considered a model system for parallel G4s, and revealed for the first time that the flexible flanking residues are recruited in a conserved and sequence-specific way, as well as unused potential for selective ligand-G4 hydrogen-bond interactions. Our results provide the true molecular basis for MycG4-targeting drugs and new critical insights into future rational design of drugs targeting MycG4 and parallel G4s that are prevalent in promoter and RNA G4s.

Oxidative Damage Induces a Vacancy G-Quadruplex That Binds Guanine Metabolites: Solution Structure of a cGMP Fill-in Vacancy G-Quadruplex in the Oxidized BLM Gene Promoter.

Guanine (G)-oxidation to 8-oxo-7,8-dihydroguanine (OG) by reactive oxygen species in genomic DNA has been implicated with various human diseases. G-quadruplex (G4)-forming sequences in gene promoters are highly susceptible to G-oxidation, which can subsequently cause gene activation. However, the underlying G4 structural changes that result from OG modifications remain poorly understood. Herein, we investigate the effect of G-oxidation on the BLM gene promoter G4. For the first time, we show that OG can induce a G-vacancy-containing G4 (vG4), which can be filled in and stabilized by guanine metabolites and derivatives. We determined the NMR solution structure of the cGMP-fill-in oxidized BLM promoter vG4. This is the first complex structure of an OG-induced vG4 from a human gene promoter sequence with a filled-in guanine metabolite. The high-resolution structure elucidates the structural features of the specific 5'-end cGMP-fill-in for the OG-induced vG4. Interestingly, the OG is removed from the G-core and becomes part of the 3'-end capping structure. A series of guanine metabolites and derivatives are evaluated for fill-in activity to the oxidation-induced vG4. Significantly, cellular guanine metabolites, such as cGMP and GTP, can bind and stabilize the OG-induced vG4, suggesting their potential regulatory role in response to oxidative damage in physiological and pathological processes. Our work thus provides exciting insights into how oxidative damage and cellular metabolites may work together through a G4-based epigenetic feature for gene regulation. Furthermore, the NMR structure can guide the rational design of small-molecule inhibitors that specifically target the oxidation-induced vG4s.

Solution Structure of Ternary Complex of Berberine Bound to a dGMP-Fill-In Vacancy G-Quadruplex Formed in the PDGFR-ß Promoter.

The G-quadruplexes (G4s) formed in the PDGFR-ß gene promoter are transcriptional modulators and amenable to small-molecule targeting. Berberine (BER), a clinically important natural isoquinoline alkaloid, has gained increasing attention due to its potential as anticancer drug. We previously showed that the PDGFR-ß gene promoter forms a unique vacancy G4 (vG4) that can be filled in and stabilized by guanine metabolites, such as dGMP. Herein, we report the high-resolution NMR structure of a ternary complex of berberine bound to the dGMP-fill-in PDGFR-ß vG4 in potassium solution. This is the first small-molecule complex structure of a fill-in vG4. This ternary complex has a 2:1:1 binding stoichiometry with a berberine molecule bound at each the 5'- and 3'-end of the 5'-dGMP-fill-in PDGFR-ß vG4. Each berberine recruits the adjacent adenine residue from the 5'- or 3'-flanking sequence to form a "quasi-triad plane" that covers the external G-tetrad of the fill-in vG4, respectively. Significantly, berberine covers and stabilizes the fill-in dGMP. The binding of berberine involves both π-stacking and electrostatic interactions, and the fill-in dGMP is covered and well-protected by berberine. The NMR structure can guide rational design of berberine analogues that target the PDGFR-ß vG4 or dGMP-fill-in vG4. Moreover, our structure provides a molecular basis for designing small-molecule guanine conjugates to target vG4s.

Evaluating Molecular Docking Software for Small Molecule Binding to G-Quadruplex DNA.

G-quadruplexes are four-stranded nucleic acid secondary structures of biological significance and have emerged as an attractive drug target. The G4 formed in the MYC promoter (MycG4) is one of the most studied small-molecule targets, and a model system for parallel structures that are prevalent in promoter DNA G4s and RNA G4s. Molecular docking has become an essential tool in structure-based drug discovery for protein targets, and is also increasingly applied to G4 DNA. However, DNA, and in particular G4, binding sites differ significantly from protein targets. Here we perform the first systematic evaluation of four commonly used docking programs (AutoDock Vina, DOCK 6, Glide, and RxDock) for G4 DNA-ligand binding pose prediction using four small molecules whose complex structures with the MycG4 have been experimentally determined in solution. The results indicate that there are considerable differences in the performance of the docking programs and that DOCK 6 with GB/SA rescoring performs better than the other programs. We found that docking accuracy is mainly limited by the scoring functions. The study shows that current docking programs should be used with caution to predict G4 DNA-small molecule binding modes.

Novel DNA Bis-Intercalator XR5944 as a Potent Anticancer Drug-Design and Mechanism of Action.

This review is dedicated to Professor William A. Denny's discovery of XR5944 (also known as MLN944). XR5944 is a DNA-targeted agent with exceptionally potent antitumor activity and a novel DNA binding mode, bis-intercalation and major groove binding, as well as a novel mechanism of action, transcription inhibition. This novel anticancer compound represents a remarkable accomplishment resulting from two decades of drug discovery by Professor Denny and coworkers. Here, we review our work on the structural study of the DNA binding mode of XR5944 and mechanistic study of XR5944 action.

PDGFR-ß Promoter Forms a Vacancy G-Quadruplex that Can Be Filled in by dGMP: Solution Structure and Molecular Recognition of Guanine Metabolites and Drugs.

Aberrant expression of PDGFR-ß is associated with a number of diseases. The G-quadruplexes (G4s) formed in PDGFR-ß gene promoter are transcriptional modulators and amenable to small molecule targeting. The major G4 formed in the PDGFR-ß gene promoter was previously shown to have a broken G-strand. Herein, we report that the PDGFR-ß gene promoter sequence forms a vacancy G-quadruplex (vG4) which can be filled in and stabilized by physiologically relevant guanine metabolites, such as dGMP, GMP, and cGMP, as well as guanine-derivative drugs. We determined the NMR structure of the dGMP-fill-in PDGFR-ß vG4 in K+ solution. This is the first structure of a guanine-metabolite-fill-in vG4 based on a human gene promoter sequence. Our structure and systematic analysis elucidate the contributions of Hoogsten hydrogen bonds, sugar, and phosphate moieties to the specific G-vacancy fill-in. Intriguingly, an equilibrium of 3'- and 5'-end vG4s is present in the PDGFR-ß promoter sequence, and dGMP favors the 5'-end fill-in. Guanine metabolites and drugs were tested and showed a conserved selectivity for the 5'-vacancy, except for cGMP. cGMP binds both the 3'- and 5'-end vG4s and forms two fill-in G4s with similar population. Significantly, guanine metabolites are involved in many physiological and pathological processes in human cells; thus, our results provide a structural basis to understand their potential regulatory functions by interaction with promoter vG4s. Moreover, the NMR structure can guide rational design of ligands that target the PDGFR-ß vG4.

Sugar Puckering Drives G-Quadruplex Refolding: Implications for V-Shaped Loops.

A DNA G-quadruplex adopting a (3+1) hybrid structure was modified in two adjacent syn positions of the antiparallel strand with anti-favoring 2'-deoxy-2'-fluoro-riboguanosine (F rG) analogues. The two substitutions promoted a structural rearrangement to a topology with the 5'-terminal G residue located in the central tetrad and the two modified residues linked by a V-shaped zero-nucleotide loop. Strikingly, whereas a sugar pucker in the preferred north domain is found for both modified nucleotides, the F rG analogue preceding the V-loop is forced to adopt the unfavored syn conformation in the new quadruplex fold. Apparently, a preferred C3'-endo sugar pucker within the V-loop architecture outweighs the propensity of the F rG analogue to adopt an anti glycosidic conformation. Refolding into a V-loop topology is likewise observed for a sequence modified at corresponding positions with two riboguanosine substitutions. In contrast, 2'-F-arabinoguanosine analogues with their favored south-east sugar conformation do not support formation of the V-loop topology. Examination of known G-quadruplexes with a V-shaped loop highlights the critical role of the sugar conformation for this distinct structural motif.

Fluorine-Mediated Editing of a G-Quadruplex Folding Pathway.

A (3+1)-hybrid-type G-quadruplex was substituted within its central tetrad by a single 2'-fluoro-modified guanosine. Driven by the anti-favoring nucleoside analogue, a novel quadruplex fold with inversion of a single G-tract and conversion of a propeller loop into a lateral loop emerges. In addition, scalar couplings across hydrogen bonds demonstrate the formation of intra- and inter-residual F⋅⋅⋅H8-C8 pseudo-hydrogen bonds within the modified quadruplexes. Alternative folding can be rationalized by the impact of fluorine on intermediate species on the basis of a kinetic partitioning mechanism. Apparently, chemical or other environmental perturbations are able to redirect folding of a quadruplex, possibly modulating its regulatory role in physiological processes.

Ligand-Induced Dimerization of a Truncated Parallel MYC G-Quadruplex.

Binding of an indoloquinoline derivative with an aminoalkyl side chain to a truncated sequence from the MYC promoter region was studied through isothermal titration calorimetry (ITC). The targeted MYC3 sequence lacks 3'-flanking nucleotides and forms a monomeric parallel quadruplex (G4) with a blunt-ended 3'-outer tetrad under the solution conditions employed. Analysis of ITC isotherms reveals multiple binding equilibria with the initial formation of a 1:2 ligand/quadruplex complex. Evaluation of electrophoretic mobilities as well as NMR spectral data confirm ligand-induced dimerization of MYC3 quadruplexes with the ligand sandwiched between the two 3'-outer tetrads. Additional ligand molecules in excess bind to the 5'-outer tetrads of the sandwich complex. Such a ligand-promoted G4 dimerization may be exploited for the controlled assembly or disassembly of G4 aggregates to expand on present quadruplex-based technologies.

DNA-RNA Hybrid Quadruplexes Reveal Interactions that Favor RNA Parallel Topologies.

A DNA G-quadruplex adopting a (3+1)-hybrid structure was substituted at its 5'-tetrad by riboguanosine (rG) analogs. Incorporation of anti-favoring rG at appropriate syn-positions of the 5'-outer tetrad induced conformational rearrangements to yield a quadruplex featuring a 5'-tetrad with reversed polarity. A high-resolution structure of a disubstituted quadruplex variant as well as direct NMR experimental evidence reveals a non-conventional C-H⋅⋅⋅O hydrogen bond in a medium groove between the 2'-OH of an rG residue adopting a C2'-endo sugar pucker and H8 of a 3'-neighboring anti-G residue. In contrast, a C3'-endo sugar conformation for another guanine ribonucleotide prevents formation of a corresponding hydrogen bond but relocates its 2'-OH substituent from the quadruplex narrow groove into a medium groove. Both the formation of favorable CHO hydrogen bridges and unfavorable interactions of the 2'-hydroxyl group in a narrow groove will promote RNA folding into a parallel topology featuring all-anti core residues and four grooves of medium size.

Selective Targeting of G-Quadruplex Structures by a Benzothiazole-Based Binding Motif.

A benzothiazole derivative was identified as potent ligand for DNA G-quadruplex structures. Fluorescence titrations revealed selective binding to quadruplexes of different topologies including parallel, antiparallel, and (3+1) hybrid structures. The parallel c-MYC sequence was found to constitute the preferred target with dissociation constants in the micromolar range. Binding of the benzothiazole-based ligand to c-MYC was structurally and thermodynamically characterized in detail by employing a comprehensive set of spectroscopic and calorimetric techniques. Job plot analyses and mass spectral data indicate noncooperative ligand binding to form complexes with 1:1 and 2:1 stoichiometries. Whereas stacking interactions are suggested by optical methods, NMR chemical shift perturbations also indicate significant rearrangements of both 5'- and 3'-flanking sequences upon ligand binding. Additional isothermal calorimetry studies yield a thermodynamic profile of the ligand-quadruplex association and reveal enthalpic contributions to be the major driving force for binding. Structural and thermodynamic information obtained in the present work provides the basis for the rational development of benzothiazole derivatives as promising quadruplex binding agents.

Observation of a Dynamic G-Tetrad Flip in Intramolecular G-Quadruplexes.

A MYC sequence forming an intramolecular G-quadruplex with a parallel topology was modified by the incorporation of 8-bromoguanosine (BrG) analogues in one of its outer G-tetrads. The propensity of the BrG analogues to adopt a syn glycosidic torsion angle results in an exceptional monomolecular quadruplex conformation featuring a complete flip of one tetrad while keeping a parallel orientation of all G-tracts as shown by circular dichroism and nuclear magnetic resonance spectroscopic studies. When substituting three of the four G-tetrad residues with BrG analogues, two coexisting quadruplex conformational isomers with an all-syn and all-anti outer G-quartet are approximately equally populated in solution. A dynamic interconversion of the two quadruplexes with an exchange rate (kex) of 0.2 s-1 is demonstrated through the observation of exchange crosspeaks in rotating frame Overhauser effect spectroscopy and nuclear Overhauser effect spectroscopy experiments at 50 °C. The kinetic properties suggest disruption of the corresponding outer G-tetrad but not of the whole quadruplex core during the tetrad flip. Conformational syn-anti isomers with homopolar and heteropolar stacking interactions are nearly isoenergetic with a transition enthalpy of 18.2 kJ/mol in favor of the all-syn isomer.

Towards the Development of Structure-Selective G-Quadruplex-Binding Indolo[3,2-b]quinolines.

The interaction of phenyl-substituted indolo[3,2-b]quinolines with DNA G-quadruplexes of different topology were studied by using a combination of spectroscopic and calorimetric methodologies. N5-Methylated indoloquinoline derivatives ((Me) PIQ) with an aminoalkyl side chain exhibit high affinities for the parallel-stranded MYC quadruplex and a (3+1)-hybrid structure combined with an excellent discrimination against the antiparallel thrombin-binding aptamer (TBA) and the human telomeric (HT) quadruplexes. Dissociation constants for the binding of the ligand to the MYC quadruplex are in the submicromolar range, being below the corresponding dissociation constants for the antiparallel-stranded quadruplexes by about one order of magnitude. Competition experiments with double-helical DNA reveal the impact of indoloquinoline structural features on the selectivity for the parallel quadruplex relative to duplex DNA. Based on a calorimetric analysis binding to MYC is shown to be equally driven by favorable enthalpic and entropic contributions with no significant impact on the type of cation present.

Sugar-Edge Interactions in a DNA-RNA G-Quadruplex: Evidence of Sequential C-H⋠⋠⋠O Hydrogen Bonds Contributing to RNA Quadruplex Folding.

DNA G-quadruplexes were systematically modified by single riboguanosine (rG) substitutions at anti-dG positions. Circular dichroism and NMR experiments confirmed the conservation of the native quadruplex topology for most of the DNA-RNA hybrid structures. Changes in the C8 NMR chemical shift of guanosines following rG substitution at their 3'-side within the quadruplex core strongly suggest the presence of C8-H⋅⋅⋅O hydrogen-bonding interactions with the O2' position of the C2'-endo ribonucleotide. A geometric analysis of reported high-resolution structures indicates that such interactions are a more general feature in RNA quadruplexes and may contribute to the observed preference for parallel topologies.

Structure and catalytic mechanism of the evolutionarily unique bacterial chalcone isomerase.

Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific (1)H/(2)H isotope exchange observed by (1)H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1.

Two subtle amino Acid changes in a transaminase substantially enhance or invert enantiopreference in cascade syntheses.

Amine transaminases (ATAs) are powerful enzymes for the stereospecific production of chiral amines. However, the synthesis of amines incorporating more than one stereocenter is still a challenge. We developed a cascade synthesis to access optically active 3-alkyl-substituted chiral amines by combining two asymmetric synthesis steps catalyzed by an enoate reductase and ATAs. The ATA wild type from Vibrio fluvialis showed only modest enantioselectivity (14 % de) in the amination of (S)-3-methylcyclohexanone, the product of the enoate-reductase-catalyzed reaction step. However, by protein engineering we created two variants with substantially improved diastereoselectivities: variant Leu56Val exhibited a higher R selectivity (66 % de) whereas the Leu56Ile substitution caused a switch in enantiopreference to furnish the S-configured diastereomer (70 % de). Addition of 30 % DMSO further improved the selectivity and facilitated the synthesis of (1R,3S)-1-amino-3-methylcyclohexane with 89 % de at 87 % conversion.