title: Structural Basis of the Substrate Range and Enantioselectivity of Two (S)-Selective ω-Transaminases.
abstract: ω-Transaminases are enzymes that can introduce an amino group in industrially interesting compounds. We determined crystal structures of two (S)-selective ω-transaminases, one from Arthrobacter sp. (Ars-ωTA) and one from Bacillus megaterium (BM-ωTA), which have 95% identical sequences but somewhat different activity profiles. Substrate profiling measurements using a range of (R)- and (S)-substrates showed that both enzymes have a preference for substrates with large, flat cyclic side groups, for which the activity of BM-ωTA is generally somewhat higher. BM-ωTA has a preference for (S)-3,3-dimethyl-2-butylamine significantly stronger than that of Ars-ωTA, as well as a weaker enantiopreference for 1-cyclopropylethylamine. The crystal structures showed that, as expected for (S)-selective transaminases, both enzymes have the typical transaminase type I fold and have spacious active sites to accommodate largish substrates. A structure of BM-ωTA with bound (R)-α-methylbenzylamine explains the enzymes' preference for (S)-substrates. Site-directed mutagenesis experiments revealed that the presence of a tyrosine, instead of a cysteine, at position 60 increases the relative activities on several small substrates. A structure of Ars-ωTA with bound l-Ala revealed that the Arg442 side chain has been repositioned to bind the l-Ala carboxylate. Compared to the arginine switch residue in other transaminases, Arg442 is shifted by six residues in the amino acid sequence, which appears to be a consequence of extra loops near the active site that narrow the entrance to the active site.
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: van Oosterwijk
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Simon
authors.initials: S
authors.lastname: Willies
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Johan
authors.initials: J
authors.lastname: Hekelaar
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Anke C
authors.initials: AC
authors.lastname: Terwisscha van Scheltinga
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Turner
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Bauke W
authors.initials: BW
authors.lastname: Dijkstra
mesh_terms: D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000637:Transaminases