A novel strategy for enzymatic synthesis of 4-hydroxyisoleucine: identification of an enzyme possessing HMKP (4-hydroxy-3-methyl-2-keto-pentanoate) aldolase activity.
A two-step enzymatic synthesis process of 4-hydroxyisoleucine is suggested. In the first step, the aldol condensation of acetaldehyde and alpha-ketobutyrate catalyzed by specific aldolase results in the formation of 4-hydroxy-3-methyl-2-keto-pentanoate (HMKP). In the second step, amination of HMKP by the branched-chain amino acid aminotransferase leads to synthesis of 4-hydroxyisoleucine. An enzyme possessing HMKP aldolase activity (asHPAL) was purified 2500-fold from a crude extract of Arthrobacter simplex strain AKU 626. Sequencing of the asHPAL structural gene showed that the purified enzyme belongs to the HpcH/HpaI aldolase family. The 4-hydroxyisoleucine was synthesized in vitro from acetaldehyde, alpha-ketobutyrate and l-glutamate using a coupled aldolase/branched-chain amino acid aminotransferase bienzymatic reaction.
Smirnov
Sergey V
SV
Ajinomoto-Genetika Research Institute, 1st Dorozhny pr. 1, Moscow, Russia. servasmi@rol.ru
Samsonova
Natalya N
NN
Novikova
Anna E
AE
Matrosov
Nikolay G
NG
Rushkevich
Natalya Y
NY
Kodera
Tomohiro
T
Ogawa
Jun
J
Yamanaka
Hiroyuki
H
Shimizu
Sakayu
S
D000079:Acetaldehyde / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002087:Butyrates / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D007532:Isoleucine / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D000637:Transaminases / Q000378:metabolism
C104672:4-hydroxyisoleucine; D001426:Bacterial Proteins; D002087:Butyrates; D004269:DNA, Bacterial; D007532:Isoleucine; D018698:Glutamic Acid; C005087:alpha-ketobutyric acid; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase; D005634:Fructose-Bisphosphate Aldolase; D000079:Acetaldehyde