No abstract

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.

The importance of amine transaminases for producing optically pure chiral precursors for pharmaceuticals and chemicals has substantially increased in recent years. The X-ray crystal structure of the (R)-selective amine transaminase from the fungus Aspergillus fumigatus was solved by S-SAD phasing to 1.84 Å resolution. The refined structure at 1.27 Å resolution provides detailed knowledge about the molecular basis of substrate recognition and conversion to facilitate protein-engineering approaches. The protein forms a homodimer and belongs to fold class IV of the pyridoxal-5'-phosphate-dependent enzymes. Both subunits contribute residues to form two active sites. The structure of the holoenzyme shows the catalytically important cofactor pyridoxal-5'-phosphate bound as an internal aldimine with the catalytically responsible amino-acid residue Lys179, as well as in its free form. A long N-terminal helix is an important feature for the stability of this fungal (R)-selective amine transaminase, but is missing in branched-chain amino-acid aminotransferases and D-amino-acid aminotransferases.

Chiral amines are important precursors for the pharmaceutical and finechemical industries. Because of this, the demand for enantiopure amines is currently increasing. Amine transaminases can produce a large spectrum of chiral amines in the (R)-or (S)-configuration, depending on their substrate scope and stereo-preference, by converting a prochiral ketone into the chiral amine while using alanine as the amine donor producing pyruvate as an a-keto acid product. In order to guide the protein engineering of transaminases to improve substrate specificity and enantioselectivity, we carried out a crystal structure analysis at 1.6A resolution of the (R)-amine transaminase from Aspergillus fumigatus with the bound inhibitor gabaculine. This revealed that Arg126 has an important role in the dual substrate recognition of this enzyme because mutating this residue to alanine reduced substantially the ability of the enzyme to use pyruvate as an amino acceptor. DatabaseCoordinates and structure factors have been deposited with the Protein Data Bank under accession code 4UUG.

Enoate reductases are versatile enzymes for the enantio‐ and regioselective addition of hydrogen to double bonds. We identified three EREDs (XenA, XenB, NemA) from Pseudomonas putida ATCC 17453 through a sequence motif search. In addition to cloning, functional expression, and biochemical characterization of these enzymes, the enoate reductases were also applied in enzyme cascade reactions in combination with a Baeyer–Villiger monooxygenase and an alcohol dehydrogenase to produce lactones.

No abstract

The (R)-selective amine transaminase from Aspergillus fumigatus was expressed in Escherichia coli and purified to homogeneity. Bright yellow crystals appeared while storing the concentrated solution in the refrigerator and belonged to space group C222 1 . X-ray diffraction data were collected to 1.27 Å resolution, as well as an anomalous data set to 1.84 Å resolution that was suitable for S-SAD phasing.

The NewProt protein engineering portal is a one-stop-shop for in silico protein engineering. It gives access to a large number of servers that compute a wide variety of protein structure characteristics supporting work on the modification of proteins through the introduction of (multiple) point mutations. The results can be inspected through multiple visualizers. The HOPE software is included to indicate mutations with possible undesired side effects. The Hotspot Wizard software is embedded for the design of mutations that modify a proteins' activity, specificity, or stability. The NewProt portal is freely accessible at http://newprot.cmbi.umcn.nl/ and http://newprot.fluidops.net/.