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Molecular design of a nylon-6 byproduct-degrading enzyme from a carboxylesterase with a beta-lactamase fold.

Kawashima Y, Ohki T, Shibata N, Higuchi Y, Wakitani Y, Matsuura Y, Nakata Y, Takeo M, Kato D, Negoro S
FEBS J 276 2547-56 (2009)
Related entries: 2e8i, 2zm0, 2zma

Cited: 8 times
EuropePMC logo PMID: 19476493

Abstract

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.

Articles citing this publication (8)

  1. Simple, yet powerful methodologies for conformational sampling of proteins. Harada R, Takano Y, Baba T, Shigeta Y. Phys Chem Chem Phys 17 6155-6173 (2015)
  2. Arabidopsis thaliana mitochondrial glyoxalase 2-1 exhibits beta-lactamase activity. Limphong P, Nimako G, Thomas PW, Fast W, Makaroff CA, Crowder MW. Biochemistry 48 8491-8493 (2009)
  3. X-ray crystallographic analysis of the 6-aminohexanoate cyclic dimer hydrolase: catalytic mechanism and evolution of an enzyme responsible for nylon-6 byproduct degradation. Yasuhira K, Shibata N, Mongami G, Uedo Y, Atsumi Y, Kawashima Y, Hibino A, Tanaka Y, Lee YH, Kato D, Takeo M, Higuchi Y, Negoro S. J Biol Chem 285 1239-1248 (2010)
  4. Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis. Negoro S, Shibata N, Tanaka Y, Yasuhira K, Shibata H, Hashimoto H, Lee YH, Oshima S, Santa R, Oshima S, Mochiji K, Goto Y, Ikegami T, Nagai K, Kato D, Takeo M, Higuchi Y. J Biol Chem 287 5079-5090 (2012)
  5. Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a beta-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolase. Ohki T, Shibata N, Higuchi Y, Kawashima Y, Takeo M, Kato D, Negoro S. Protein Sci 18 1662-1673 (2009)
  6. On the induced-fit mechanism of substrate-enzyme binding structures of nylon-oligomer hydrolase. Baba T, Harada R, Nakano M, Shigeta Y. J Comput Chem 35 1240-1247 (2014)
  7. Unraveling the degradation of artificial amide bonds in nylon oligomer hydrolase: from induced-fit to acylation processes. Baba T, Boero M, Kamiya K, Ando H, Negoro S, Nakano M, Shigeta Y. Phys Chem Chem Phys 17 4492-4504 (2015)
  8. Letter Mutations affecting the internal equilibrium of the reaction catalyzed by 6-aminohexanoate-dimer hydrolase. Negoro S, Kawashima Y, Shibata N, Kobayashi T, Baba T, Lee YH, Kamiya K, Shigeta Y, Nagai K, Takehara I, Kato D, Takeo M, Higuchi Y. FEBS Lett 590 3133-3143 (2016)


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