Structure Report for: 3VIS

Kitadokoro, K., Thumarat, U., Nakamura, R., Nishimura, K., Karatani, H., Suzuki, H., Kawai, F.

Title: Crystal structure of cutinase Est119 from Thermobifida alba AHK119 that can degrade modified polyethylene terephthalate at 1.76A resolution
Kitadokoro K, Thumarat U, Nakamura R, Nishimura K, Karatani H, Suzuki H, Kawai F
Ref: Polymer Degradation and Stability, 97:771, 2012 : PubMed
Abstract


ESTHER: Kitadokoro_2012_Polym.Degrad.Stab_97_771
PubMedSearch: Kitadokoro 2012 Polym.Degrad.Stab 97 771
Gene_locus related to this paper: 9acto-f7ix06

Abstract

We determined the crystal structure of a cutinase from Thermobifida alba AHK119 (Est119) at a resolution of 1.76A. The overall structure of Est119 displays a typical alpha/beta-hydrolase fold consisting of a central twisted beta-sheet of nine beta-strands that are flanked by nine alpha-helices on both sides. The refined model contains two monomers in the asymmetric unit that form a dimer interface; a polyethylene glycol fragment is bound in the interface. Polyethylene glycol-binding site on the protein may suggest a glycol-binding site. A putative polymer-recognizing groove is observed to continue through the catalytic pocket. Water molecules are bound to hydrophilic amino acids along the groove, indicating the alternating pattern of polar and hydrophobic residues.



        

Title: Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119
Thumarat U, Nakamura R, Kawabata T, Suzuki H, Kawai F
Ref: Applied Microbiology & Biotechnology, 95:419, 2012 : PubMed
Abstract


ESTHER: Thumarat_2012_Appl.Microbiol.Biotechnol_95_419
PubMedSearch: Thumarat 2012 Appl.Microbiol.Biotechnol 95 419
PubMedID: 22183084
Gene_locus related to this paper: 9acto-f7ix06

Abstract

Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 degrees C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an alpha/beta-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second beta-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca(2+) and was stable up to 60 degrees C with 150 mM Ca(2+). Another identical gene was located in tandem in the upstream of est119.