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PDBsum entry 2zma
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References listed in PDB file
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Key reference
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Title
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Molecular design of a nylon-6 byproduct-Degrading enzyme from a carboxylesterase with a beta-Lactamase fold.
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Authors
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Y.Kawashima,
T.Ohki,
N.Shibata,
Y.Higuchi,
Y.Wakitani,
Y.Matsuura,
Y.Nakata,
M.Takeo,
D.Kato,
S.Negoro.
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Ref.
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Febs J, 2009,
276,
2547-2556.
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PubMed id
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Abstract
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A carboxylesterase with a beta-lactamase fold from Arthrobacter possesses a low
level of hydrolytic activity (0.023 mumol.min(-1).mg(-1)) when acting on a
6-aminohexanoate linear dimer byproduct of the nylon-6 industry (Ald).
G181D/H266N/D370Y triple mutations in the parental esterase increased the
Ald-hydrolytic activity 160-fold. Kinetic studies showed that the triple mutant
possesses higher affinity for the substrate Ald (K(m) = 2.0 mm) than the
wild-type Ald hydrolase from Arthrobacter (K(m) = 21 mm). In addition, the
k(cat)/K(m) of the mutant (1.58 s(-1).mm(-1)) was superior to that of the
wild-type enzyme (0.43 s(-1).mm(-1)), demonstrating that the mutant efficiently
converts the unnatural amide compounds even at low substrate concentrations, and
potentially possesses an advantage for biotechnological applications. X-ray
crystallographic analyses of the G181D/H266N/D370Y enzyme and the inactive
S112A-mutant-Ald complex revealed that Ald binding induces rotation of
Tyr370/His375, movement of the loop region (N167-V177), and flip-flop of Tyr170,
resulting in the transition from open to closed forms. From the comparison of
the three-dimensional structures of various mutant enzymes and site-directed
mutagenesis at positions 266 and 370, we now conclude that Asn266 makes suitable
contacts with Ald and improves the electrostatic environment at the N-terminal
region of Ald cooperatively with Asp181, and that Tyr370 stabilizes Ald binding
by hydrogen-bonding/hydrophobic interactions at the C-terminal region of Ald.
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