Second‐Generation Engineering of a Thermostable Transketolase (TK_Gst) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity

Abstract

The transketolase from Geobacillus stearothermophilus (TK ) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non-α-hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TK based on double-site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TK for various applications in asymmetric carboligation.

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

“…Comparison of the results for the here tested Ec TK variants with variants of Gst TK described by Zhou et al . shows that effects of amino acid exchanges can also differ for TKs from different organisms.…”

“…Further, H26 in Ec TK was identified as key residue to influence the stereoselectivity . Among further residues, which were identified to influence stereoselectivity, is phenylalanine in standard position 434 found in Ec TK, Gst TK and Sc TK and conserved with 62 % in the whole TK superfamily . Also leucine in standard position 382 is conserved among these TKs and found in 61 % of all sequences of the TK superfamily.…”

“…Among further residues, which were identified to influence stereoselectivity, is phenylalanine in standard position 434 found in Ec TK, Gst TK and Sc TK and conserved with 62 % in the whole TK superfamily . Also leucine in standard position 382 is conserved among these TKs and found in 61 % of all sequences of the TK superfamily. Besides, an isoleucine found in Ec TK and Sc TK in standard position 189 is replaced by L191 in Gst TK .…”

“…Also leucine in standard position 382 is conserved among these TKs and found in 61 % of all sequences of the TK superfamily. Besides, an isoleucine found in Ec TK and Sc TK in standard position 189 is replaced by L191 in Gst TK . Aspartate in standard position 469 plays a role in substrate binding, activity enhancement and stereoselectivity, is conserved among the three TKs mentioned above and found in 59 % of TK sequences.…”

“…Especially the exchange L191V resulted in R ‐selective Gst TK variants with ee ‐values up to 84 % (see SI Table S1). Effects on the orientation of the acceptor propanal were discussed . In Ec TK an isoleucine residue is found at standard position 189 (SI Table S2).…”