l-Threonine aldolase (LTA)
is an attractive tool in organic
chemistry for catalyzing the formation of β-hydroxy-α-amino
acids with two chiral centers. The enzyme has a strict selectivity
for Cα of β-hydroxy-α-amino acids but
a moderate selectivity for Cβ
, limiting
its wide applications in stereospecific carbon–carbon bond
synthesis. Here, a combinatorial active-site saturation test/iterative
saturation mutagenesis (CAST/ISM) strategy was applied to accelerate
directed evolution of LTA in diastereoselectivity. A total of 27 amino
acid residues lining the substrate pocket were selected and divided
into two groups based on their functional region. In silico screening and site-directed saturation mutagenesis identified six
(3 + 3) amino acid residues of them with a significant effect on diastereoselectivity.
The ISM strategy was then performed in and between each group to obtain
the best combinatorial mutation. As a result, a variant RS1 (Y8H/Y31H/I143R/N305R)
was obtained with a dramatically improved preference for the synthesis
of l-syn-3-[4-(methylsulfonyl)phenylserine].
The product with a de value of 99.5% (73.2% conv) was produced
in a 20 L reactor, which is promising in the industrial synthesis
of aromatic l-syn-β-hydroxy-α-amino
acids with LTA. The variant also represented a significant selective
improvement to other l-phenylserine derivatives. The de values
of 2-NO2-, 4-NO2-, H-, and 4-CH4-substituted l-phenylserine derivatives were more than 99%
syn
by dynamic control. The insight of the mutant model suggests
that the binding pocket of the active center was reshaped.
Diastereoselectivity
of l-threonine aldolase (LTA) was
determined by paths of aldehydes attacking a pyridoxal phosphate–glycine
complex. Thus, strategies of enhancing the syn path
and blocking the anti path were performed to modify
LTA. A mutant (Y31H/N305R) was constructed with a substrate preference
increase from 3.32 to 42.04. Medium engineering was investigated.
Consequently, the de value of l-syn-3-[4-(methylsulfonyl)phenylserine]
reached 93.1% (87.2%conv). The study clarified the factors
affecting diastereoselectivity of LTA and provided a theorem for rational
modification of LTA’s diastereoselectivity.
Abundance of bundle sheath organelles in the heavy salt Four ecotypes of Phragmites australis from different habitats in northwest China were examined to compare their photosyn-meadow ecotype was intermediate. The swamp ecotype had photosynthetic enzyme activities typical of C 3 type plants, thetic characteristics. In a swamp ecotype, the 13 C value of leaf materials was −34.0‰, and bundle sheath cells con-whereas the dune ecotype had an increased activity of phosphoenolpyruvate carboxylase (PEPC), a key C 4 enzyme, and tained a small amount of organelles and round-shaped chloroplasts, as being similar to typical C 3 plants. In a dune ecotype, a decreased ribulose 1,5-bisphosphate carboxylase (Rubisco) activity. The light salt meadow and heavy salt meadow eco-the 13 C value was −20.9‰ and bundle sheath cells contypes had substantial activities of PEPC, which indicates tained oval-shaped chloroplasts with poorly-developed grana. potential for C 4 photosynthesis. These data suggest that this In light and heavy salt meadow ecotypes, 13 C values were −30.6‰ and −35.6‰, respectively. The shape of bundle species evolved the C 3 -like ecotype in swamp environments sheath chloroplasts in the light salt meadow ecotype was and the C 4 -like C 3 -C 4 intermediate in dune desert environintermediate between those of the swamp and dune ecotypes. ments, and C 3 -like C 3 -C 4 intermediates in salt environments.
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.
The L-threonine aldolase from Leishmania major was engineered to improve diastereoselectivity by a CAST/ISM strategy, providing insights into the relationship between physico -chemical properties of substrate access path and diastereoselectivity....
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