Cβ-Selective Aldol Addition of d-Threonine Aldolase by Spatial Constraint of Aldehyde Binding

Abstract

d-Threonine aldolase (DTA) is a useful biocatalyst that reversibly converts glycine and aldehyde to β-hydroxy-α-d-amino acid. However, low activity and poor diastereoselectivity limit its applications. Here we report DTA from Filomicrobium marinum (FmDTA) that shows much higher activity and Cβ-stereoselectivity in d-threonine production compared with those of other known DTAs. We determine the FmDTA structure at a 2.2 Å resolution and propose a DTA catalytic mechanism with a kernel of the Lys49 inner proton sink and metal ion in the aldol reaction cycle. The enzyme is rationally engineered to have high Cβ-stereoselectivity based on spatial constraint at the anti-specific aldehyde position in the mechanism, and the rational strategy is further applied to other DTAs for syn-production. The final FmDTAG179A/S312A variant exhibits a near-perfect 99.5% de value for d-threonine and maintains the de value above 93% even under kinetically unfavorable conditions. This study demonstrates how a detailed understanding of the reaction mechanism can be used for rational protein engineering.

Structure of pyridoxal 5′-phosphate-bound D-threonine aldolase from Chlamydomonas reinhardtii

“…In fold-type III enzymes, the pyridine N atom is known to interact with various types of bulky and nonacidic residues, and in bacterial alanine racemase (Shaw et al, 1997) and AxDTA (Uhl et al, 2015) it interacts with arginine and glutamine, respectively, which cannot donate a proton. In the suggested reaction mechanism, it has been reported that both LTA and DTA form quinonoid intermediates (di Salvo et al, 2014;Uhl et al, 2015;Park et al, 2021). However, we propose that the quinonoid intermediate is not formed by CrDTA and AxDTA because the pyridine N atom of these DTAs cannot be protonated by the interacting Gln residue.…”

“…3b; PDB entry 7dib). FmDTA is characterized by higher K m values for d-threonine and d-allothreonine (23 and 20 mM, respectively) than other DTAs (Table 2; Park et al, 2021). FmDTA also has a similar active site to those of CrDTA and AxDTA.…”

“…Uhl et al (2015) predicted His193 to be the catalytic residue in AxDTA and proposed that the -hydroxy group of the substrate would replace the water molecule of the metal ion and draw a proton from the -hydroxy group using the histidine residue. However, in a recent study Park et al (2021) prepared and analyzed an H183A mutant of the putative catalytic residue and reported that the mutant had approximately 10% of the activity of the wild type. This indicates that the histidine residue is not an essential catalytic residue and the authors proposed a new reaction mechanism using the side chain of Lys49 (corresponding to Lys80 in CrDTA), which forms a Schiff base with PLP, as a new catalytic residue.…”

“…The structural similarity of CrDTA to known enzymes was explored using DALI (Holm, 2022). AxDTA (PDB entry 4v15; Uhl et al, 2015) and FmDTA (PDB entry 7dib; Park et al, 2021) have high structural similarity to CrDTA (AxDTA, Z-score 51.0, r.m.s.d. 1.7 A ˚and 44% sequence identity for 367 C pairs; FmDTA, Z-score 50.9, r.m.s.d.…”

Substrate access path-guided engineering of l-threonine aldolase for improving diastereoselectivity

“…[4][5][6] b-Hydroxy-aamino acids are valuable chiral building blocks for pharmaceuticals such as florfenicol, droxidopa, thiamphenicol and chloramphenicol. [7][8][9][10] LTA has a strict selectivity for C a of b-hydroxy-aamino acids with a value of enantiomeric excess (ee) over 99.0%. However, the selectivity for C b is moderate, limiting its industrial applications.…”