title: Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626.
abstract: Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, alpha-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM alpha-ketobutyrate, and 100 mM L-glutamate with a molar yield to alpha-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl(2) x 4H(2)O at 28 degrees C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and alpha-ketobutyrate was purified from A. simplex AKU 626. Mn(2+) and pyridoxal 5'-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ogawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamanaka
authors.forename: Junichi
authors.initials: J
authors.lastname: Mano
authors.forename: Yuko
authors.initials: Y
authors.lastname: Doi
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Horinouchi
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kodera
authors.forename: Noriki
authors.initials: N
authors.lastname: Nio
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Smirnov
authors.forename: Natalya N
authors.initials: NN
authors.lastname: Samsonova
authors.forename: Yury I
authors.initials: YI
authors.lastname: Kozlov
authors.forename: Sakayu
authors.initials: S
authors.lastname: Shimizu
mesh_terms: D001173:Arthrobacter / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000502:physiology*; D007532:Isoleucine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000637:Transaminases / Q000502:physiology*
chemical_list: C104672:4-hydroxyisoleucine; D007532:Isoleucine; D000637:Transaminases; D005634:Fructose-Bisphosphate Aldolase