An Aminocaprolactam Racemase from Ochrobactrum anthropi with Promiscuous Amino Acid Ester Racemase Activity

Abstract

The kinetic resolution of amino acid esters (AAEs) is a useful synthetic strategy for the preparation of single-enantiomer amino acids. The development of an enzymatic dynamic kinetic resolution (DKR) process for AAEs, which would give a theoretical yield of 100 % of the enantiopure product, would require an amino acid ester racemase (AAER); however, no such enzyme has been described. We have identified low AAER activity of 15 U mg in a homologue of a PLP-dependent α-amino ϵ-caprolactam racemase (ACLR) from Ochrobactrum anthropi. We have determined the structure of this enzyme, OaACLR, to a resolution of 1.87 Å and, by using structure-guided saturation mutagenesis, in combination with a colorimetric screen for AAER activity, we have identified a mutant, L293C, in which the promiscuous AAER activity of this enzyme towards l-phenylalanine methyl ester is improved 3.7-fold.

Overview on Multienzymatic Cascades for the Production of Non-canonical α-Amino Acids

“…Remarkably, RoNHase and ACLR were reported to suffer inhibition by the substrate α-amino nitrile, and thus, substrate concentrations need to be taken into account if using this MEC combination (Yasukawa and Asano, 2012). A recent study has showed that the ACLR from Ochrobactrum anthropi also racemizes α-AA esters (Frese et al, 2018). This activity opens up new enzyme combinations for the synthesis of enantiopure α-AAs, coupling ACLR with stereospecific esterases (see section "AAER/Esterase System").…”

“…L2HAO: L-2-hydroxy acid oxidase (e.g., AVLOX from Aerococcus viridans); TPL: WT or engineered tyrosine phenol lyase from Symbiobacterium toebii (Li et al, 2020). (F) Theoretical approach for the deracemization of AA ester racemates using an "amino acid ester racemase" (AAER, an ACLR homolog) and an enantioselective esterase (Frese et al, 2018). (G) 5-Hydroxy-tryptophan synthesis through a L-phenylalanine 4-hydroxylase coupled to cofactor regeneration.…”

“…Recently, the deracemization of AA esters through the action of a combination of an AA-ester racemase (AAER) and an enantioselective esterase has been proposed ( Frese et al, 2018 ). Different PLP-dependent ACLR-homologs (see section “Amidase Process”) with AAER activity on phenylalanineamide were identified, thus allowing to propose a putative AAER/esterase system for deracemization of AA esters, analogous to the “Amidase Process” ( Figure 12F ).…”