A new target region for changing the substrate specificity of amine transaminases

Abstract

(R)-stereospecific amine transaminases (R-ATAs) are important biocatalysts for the production of (R)-amine compounds in a strict stereospecific manner. An improved R-ATA, ATA-117-Rd11, was successfully engineered for the manufacture of sitagliptin, a widely used therapeutic agent for type-2 diabetes. The effects of the individual mutations, however, have not yet been demonstrated due to the lack of experimentally determined structural information. Here we describe three crystal structures of the first isolated R-ATA, its G136F mutant and engineered ATA-117-Rd11, which indicated that the mutation introduced into the 136th residue altered the conformation of a loop next to the active site, resulting in a substrate-binding site with drastically modified volume, shape, and surface properties, to accommodate the large pro-sitagliptin ketone. Our findings provide a detailed explanation of the previously reported molecular engineering of ATA-117-Rd11 and propose that the loop near the active site is a new target for the rational design to change the substrate specificity of ATAs.

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.…”

“…This loop changes its conformation from a closed to an open form upon binding of the inhibitor gabaculin in the ( R )-ATA of A. fumigatus 35 thereby moving R126 out of the active site, and due to high B-factors of the amino acid residues the loop is also expected to be flexible in AT-ωTA of A. terreus 15. Moreover, an altered conformation of the respective loop in ATA-117-Rd11 variant compared to its wildtype might be one of the reasons for its ability to accommodate the pro-sitagliptin ketone13. While the exact position of R138 in the loop of ATA-117-Rd11 is not conserved compared to other published ( R )-ATA structures, the guanidinium groups of all loop arginines localise in a similar position.…”

“…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. The sequences were aligned using ClustalW254 and the phylogenetic tree was visualised using the programme Archaeopteryx55.…”

“…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.…”

Substrate screening of amino transaminase for the synthesis of a sitagliptin intermediate

“…16 The enzymatic binding pocket can be divided into a larger pocket and a smaller one. 16 Considering the ester part of the substrates should be accommodated in the larger one to ensure satisfactory stereoselectivity, 16,19 some large substituents of the ester, such as benzyl, phenylethyl, and cyclohexyl groups, were chosen. Some small substituents such as the methyl, ethyl, and propyl groups were also chosen to fully map the substrate profile of the enzyme.…”