Mutability‐Landscape‐Guided Engineering of l‐Threonine Aldolase Revealing the Prelog Rule in Mediating Diastereoselectivity of C−C Bond Formation

Abstract

l‐threonine aldolase (LTA) catalyzes C−C bond synthesis with moderate diastereoselectivity. In this study, with LTA from Cellulosilyticum sp (CpLTA) as an object, a mutability landscape was first constructed by performing saturation mutagenesis at substrate access tunnel amino acids. The combinatorial active‐site saturation test/iterative saturation mutation (CAST/ISM) strategy was then used to tune diastereoselectivity. As a result, the diastereoselectivity of mutant H305L/Y8H/V143R was improved from 37.2 %syn to 99.4 %syn. Furthermore, the diastereoselectivity of mutant H305Y/Y8I/W307E was inverted to 97.2 %anti. Based on insight provided by molecular dynamics simulations and coevolution analysis, the Prelog rule was employed to illustrate the diastereoselectivity regulation mechanism of LTA, holding that the asymmetric formation of the C−C bond was caused by electrons attacking the carbonyl carbon atom of the substrate aldehyde from the re or si face. The study would be useful to expand LTA applications and guide engineering of other C−C bond‐forming enzymes.

Discovery and Engineering of the l-Threonine Aldolase from Neptunomonas marine for the Efficient Synthesis of β-Hydroxy-α-amino Acids via C–C Formation

“…We further compared the diastereoselective mechanisms of Nm LTA with two other LTAs, namely, Cp LTA ( Cellulosilyticum sp) and Pp LTA . Notably, Cp LTA has been previously found to contain two different substrate binding channels that govern the enzyme diastereoselectivity.…”

“…Among these, Nm LTA exhibited the highest de value of 89.5% and an efficient enzymatic activity (64.8 U mg –1 ) that was ∼1.4-fold higher than the highest native LTA reported so far (Figure B and Table ). Furthermore, only one pair of diastereomeric products, l - threo -MTPS and l - erythro -MTPS, were detected in the aldol reaction catalyzed by Nm LTA (Figure S2). Further analyses showed that Nm LTA had a maximum enzymatic activity at 30 °C and pH 8.0, and the efficiency could be further improved by increasing the PLP concentration (Figure S3).…”

“…Taken together, our results proved that cavity B was unlikely a binding pocket for 4-MTB and does not participate in regulating the diastereoselectivity of NmLTA, which supports our "dual-conformation" mechanism. This work therefore rules out the formerly proposed "two substrate pathways" mechanism as observed in other LTA, 33,34,36 and proposes a novel diastereoselectivity mechanism for NmLTA. Interestingly, a similar mechanism was also observed in a pyruvate-dependent aldolase, where the pyruvate enolate could attack the si or re face of the substrate succinic semialdehyde to yield racemic products.…”

“…Several groups have designed a number of LTA variants with improved stereoselectivity at the C β -site based on these crystal structures or homologue models. ,,− For example, Lin′s group increased the de value of Actinocorallia herbida LTA ( Ah LTA) from 58 to 81% for l - threo -MTPS by modulating the substrate binding pocket, and they further enhanced the Ah LTA de value to 93.7% via rational design by combining molecular dynamics simulations and free-energy calculations. , Cai et al improved the diastereoselectivity of Pseudomonas putida LTA ( Pp LTA) from 32 to 85% for l - threo -4-nitrophenylserine via computer-aided directed evolution . In addition, Zheng and colleagues conducted a series of structure-based designs of LTA variants to produce an enhanced de value up to 99% for l - threo -MTPS. , This group also proposed that the substrate 4-methylsulfonylbenzaldehyde (4-MTB) could potentially bind to LTA along two distinct channels, enabling in the formation of two diastereomeric products. − …”

Rational design of enzyme activity and enantioselectivity

“…Computational design based on calculation of binding energy was also applied to design enzyme variants with improved diastereoselectivity. It was assumed that for the L-threonine aldolase (LTA) from Bacillus nealsonii ( Bn LTA), the syn -configuration was formed by substrate 4-methylsulfonyl benzaldehyde (MTB) attack of the α-carbon atom of pyridoxal phosphate (PLP)−glycine through the syn path, and the anti -configuration was formed by attack of the α-carbon atom of PLP−glycine through the anti path ( Zheng et al, 2020 ; Zheng et al, 2023 ). Hence, in order to improve diastereoselectivity of Bn LTA towards syn -configuration product, the variants in syn -path with improved binding affinity to substrate and the variants in anti -path with reduced binding affinity to substrate were virtually screened by calculating binding energy using tool of Discovery Studio.…”