Extended reaction scope of thiamine diphosphate dependent cyclohexane-1,2-dione hydrolase: from C-C bond cleavage to C-C bond ligation.
ThDP-dependent cyclohexane-1,2-dione hydrolase (CDH) catalyzes the CC bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate. One of the two reactivities of CDH was selectively knocked down by mutation experiments. CDH-H28A is much less able to catalyze the CC bond formation, while the ability for CC bond cleavage is still intact. The double variant CDH-H28A/N484A shows the opposite behavior and catalyzes the addition of pyruvate to cyclohexane-1,2-dione, resulting in the formation of a tertiary alcohol. Several acyloins of tertiary alcohols are formed with 54-94 % enantiomeric excess. In addition to pyruvate, methyl pyruvate and butane-2,3-dione are alternative donor substrates for CC bond formation. Thus, the very rare aldehyde-ketone cross-benzoin reaction has been solved by design of an enzyme variant.
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104 Freiburg (Germany).
Sabrina
S
Loschonsky
Tobias
T
Wacker
Simon
S
Waltzer
Pier Paolo
PP
Giovannini
Michael J
MJ
McLeish
Susana L A
SL
Andrade
Michael
M
Müller
D019943:Amino Acid Substitution; D020627:Azoarcus / Q000201:enzymology; D001573:Benzoin / Q000737:chemistry; D055162:Biocatalysis; D002244:Carbon / Q000737:chemistry; D020134:Catalytic Domain; D003512:Cyclohexanones / Q000737:chemistry / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000737:chemistry / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism
D003512:Cyclohexanones; D002244:Carbon; C018031:1,2-cyclohexanedione; D019289:Pyruvic Acid; D006867:Hydrolases; D001573:Benzoin; D013835:Thiamine Pyrophosphate
asymmetric synthesis; benzoin condensation; enzyme catalysis; protein engineering; tertiary alcohols