The substrate specificity, enantioselectivity and structure of the (R)‐selective amine : pyruvate transaminase from Nectria haematococca

Abstract

During the last decade the use of transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. Transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (S)-enantiospecificity. Transaminases with (R)-specificity are needed to expand the applications of these enzymes in biocatalysis. In this work we have identified a fungal putative (R)-specific transaminase from the Eurotiomycetes Nectria haematococca, cloned a synthetic version of this gene, demonstrated (R)-selective deamination of several substrates including (R)-α-methylbenzylamine, as well as production of (R)-amines, and determined its crystal structure. The crystal structures of the holoenzyme and the complex with an inhibitor gabaculine offer the first detailed insight into the structural basis for substrate specificity and enantioselectivity of the industrially important class of (R)-selective amine : pyruvate transaminases.DatabaseThe atomic coordinates and structure factors for the Nectria TAm in holoenzyme and gabaculine-bound forms have been deposited in the PDB as entries 4cmd and 4cmf respectively.Structured digital abstract• TAm and TAm bind by x-ray crystallography (View interaction)

Discovery and structural characterisation of new fold type IV-transaminases exemplify the diversity of this enzyme fold

“…Interestingly, a flexible loop (e.g. aa K125-I135 in AT-ωTA), which is present in ( R )-ATAs13151735 as part of the active site (the loop of chain B as part of the active site of chain A and vice versa containing an arginine residue, which is discussed to play a role in dual substrate recognition of ( R )-ATAs131535) is significantly shorter in Cpu TA1 (aa E125-G128) and does not contain any positively charged amino acid. Thus, the loop is located further away from the PLP binding site creating more space in the active site (Fig.…”

“…D-ATAs: 1DAA from Bacillus sp YM-131, 4M0J from Burkholderia thailandensis E264; R-ATAs: 4CE5 from Aspergillus terreus 15, 4CHI from Aspergillus fumigatus 18, 4CMD from Nectria haematococca 17, 3WWH from Arthrobacter sp. KNK16813; BCATs: 1IYE from E. coli 32, 2EIY from Thermus thermophilus , 3HT5 from Mycobacterium tuberculosis 47, 3JZ6 from Mycobacterium smegmatis 48, 3UYY from Deionococcus radiodurans 49, 3U0G from Burkholderia pseudomallei , 4DQN from Streptococcus mutans 50, 2COI36 and 2HG851 from human; ADCLs: 1I2K from E. coli , 2Y4R from Pseudomonas aeruginosa 52, and a few putative aminotransferases: 2ZGI from Thermus thermophilus HB853, 3CEB from Haemophilus somnus , 3CSW from Thermotoga maritima , 3LUL from Legionella pneumophila , 3QQM from Mesorhizobium loti, 3SNO from Corynebacterium glutamicum , 4JXU from Sinorhizobium meliloti and 4TVI from Brucella abortus.…”

“…Although a lot of effort has already been made to improve and tailor transaminases according to industrial requirements by protein, substrate and reaction engineering89101112, there is still a clear need for further enhancement of known transaminases or the discovery of new, especially ( R )-selective transaminases (( R )-ATA) exhibiting improved properties. All ( R )-amine transaminases described in the literature so far are members of the fold type IV PLP-dependent enzyme family131415161718. This family includes d -amino acid aminotransferases, L-branched chain aminotransferases and 4-amino-4-deoxychorismate lyases (ADCL)19.…”

A subfamily of PLP-dependent enzymes specialized in handling terminal amines

“…For example 2-aminoethylphosphonate-pyruvate AT (belonging to fold-type I, but not to AT-II; [26]) acts on a substrate that definitely qualifies as an ω-amine. Several ω-transaminases have also been identified belonging to fold-type IV; these enzymes are being used (much like some belonging to the AT-II subgroup; see section below) for both the asymmetric synthesis and resolution of chiral amines [18,27].…”

Transaminases and their Applications

“…In the last years various transaminases with (R)-enantiopreference have been discovered. A study has established the structure of an (R)-selective amine-pyruvate aminotransferase from Nectria haematococca, which has the ability to convert (R)-methylbenzylamine and several (R)-amines and ketones [27]. Furthermore (R)-selective amine transaminases from A. terreus and Aspergillus fumigatus have been characterized [14,28].…”