���� JFIF ` ` �� C MHETase contains five disulfide bonds (Fig. F.L.K. - TRAM 2 CADAM 3A). MHETase and the C. thiooxydans and Hydrogenophaga sp. The presence of confirmed MHETase homologs in C. thiooxydans and Hydrogenophaga sp. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. Namely, overall degradation scales with PETase loading within the tested range (0 to 2.0 mg PETase/g PET), but the inclusion of MHETase in the degradation reaction markedly improves depolymerization and this synergistic enhancement also scales with MHETase loading. For example, as observed in fungal cellulase systems for cellulose depolymerization, these mixtures typically contain a subset of enzymes to act directly on solid polymeric substrates via interfacial enzyme mechanisms, and complementary enzymes (e.g., β-glucosidases) that further process solubilized intermediates to monomeric constituents (e.g., cellobiose hydrolysis to glucose). Enter multiple addresses on separate lines or separate them with commas. !��FDS ��@Ƀ��\�b��V�㲓��PӸ���yb|��k_o0��a[���t}Z�p��(i��.�?습@�˳�q��7��rfN*��s07�_`y�Į榨v�#�Ռ����P'X����ށk���1"�&�b˩C�ɬ�8@�(�9"]+z�~ to�z�q���LcϘ�t-x�{8�b���K��g.9,�s�\����ᗺ"�G���?��|���l�cL=� =��M�[���Q�G�. This title can be used by several travelers. However, MHETase is also the most susceptible to substrate inhibition with a Kk value of 307.30 ± 20.65 µM. We do not capture any email address. Drawing inspiration from our structural analyses, this complementary study offers further insights into the two-enzyme PETase/MHETase system. 3E). cross-cutting themes. (except special services). Two-enzyme systems for complete PET degradation have been examined previously, either derived from a single microorganism (e.g., Thermobifida fusca) (58) or screened from multiple sources for optimal activity (25, 59). endstream Deconstruction of recalcitrant polymers, such as cellulose or chitin, is accomplished in nature by synergistic enzyme cocktails that evolved over millions of years. As in acylation, metastable configurations along the MFEP are not observed. E6, they are primarily able to consume soluble, xenobiotic intermediates. Published by PNAS. %���� We observed that residue Phe415 adopts a “closed” orientation on substrate binding consistent with prior substrate bound structures (PDB ID codes 6QGA and 6QGB) (35), and the partially occupied site results in an intermediate dual “open/closed” conformation (SI Appendix, Fig. PML113 suggests that these bacteria may harbor abilities for TPA catabolism (SI Appendix, Fig. Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF. To elucidate sequence relationships between MHETase and tannase family enzymes, we performed bioinformatic analyses of 6,671 tannase family sequences retrieved from the National Center for Biotechnology Information (NCBI) via PSI-BLAST (50). This work was performed as part of the Bio-Optimized Technologies to keep Thermoplastics out of Landfills and the Environment Consortium and was supported by the Advanced Manufacturing Office and Bioenergy Technologies Office under contract DE-AC36-08GO28308 with the NREL, operated by Alliance for Sustainable Energy, LLC. In fact, the terminal residues of the lid domain converge to within hydrogen-bonding distance of each other (Tyr252-Ala469, 2.9 Å), creating a compact linkage to the hydrolase domain. Author contributions: G.T.B. A small fossil reptile related to dinosaurs and pterosaurs suggests a miniaturized origin for some of the largest animals to live on Earth. Additional simulation details are in SI Appendix, Supplementary Materials and Methods. designed research; B.C.K., E.E., M.D.A., J.E.G., R.G., F.L.K., I.P., E.T., J.J.A., H.P.A., G.D., C.W.J., N.A.R., C.J.S., V.C., C.M.P., H.L.W., and B.S.D. Serine hydrolases catalyze a two-step reaction involving formation of an acyl-enzyme intermediate (acylation) that is released hydrolytically in the second step (deacylation) (34). Main-chain atoms of the linkage residues Tyr252 and Ala469 are colored lime green (also in B). Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. In one simulation, EG initially maintains a hydrogen bond with His528 for ∼100 ps, then dislodges from the active site, and is free in solution within 1 ns.