Polyethylene terephthalate (PET) is among the most extensively produced plastics, but huge amounts of PET wastes that have accumulated in the environment have become a serious threat to the ecosystem. Applying PET hydrolytic enzymes to depolymerize PET is an attractive measure to manage PET pollution, and searching for more effective enzymes is a prerequisite to achieve this goal. A thermostable cutinase that originates from the leaf-branch compost termed ICCG is the most effective PET hydrolase reported so far. Here, we illustrated the crystal structure of ICCG in complex with the PET analogue, mono(2-hydroxyethyl)terephthalic acid, to reveal the enzyme–substrate interaction network. Furthermore, we applied structure-based engineering to modify ICCG and screened for variants that exhibit higher efficacy than the parental enzyme. As a result, several variants with the measured melting temperature approaching 99 °C and elevated PET hydrolytic activity were obtained. Finally, crystallographic analyses were performed to reveal the structural stabilization effects mediated by the introduced mutations. These results are of importance in the context of understanding the mechanism of action of the thermostable PET hydrolytic enzyme and shall be beneficial to the development of PET biodegradation platforms.
Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.
Isoprenyl diphosphate synthases (IDSs) catalyze condensation reactions of isoprene units to produce isoprenoids or terpenoids, the largest class of natural products on earth. IDSs are divided into trans-and cis-IDS superfamilies depending on the configuration of the resulting carbon−carbon double bonds. Compared with trans-IDSs, cis-IDS family members exhibit more variable active site structure and versatile function. The archetypal cis-IDSs are homodimers and produce linear isoprenoids via headto-tail condensation. Recently, heterodimeric cis-IDSs containing a noncatalytic subunit that also belongs to the cis-IDS superfamily have been identified. Moreover, several cis-IDS-fold members have been found to produce nonlinear isoprenoids via "head-to-middle" condensation. In this Review, we summarize the structural features and catalytic mechanism of the versatile cis-IDS superfamily. This information provides important guidance for research regarding biosynthesis of important natural products and medical intermediates, cellular metabolism, and disease control.
Many steroids are important pharmaceutically active compounds, while cytochrome P450 monooxygenases (CYPs) are attractive enzymes for applications in steroidal drug synthesis. However, the catalytic efficiency of existing P450s is not routinely high enough, as well as the molecular basis for selectivity control is unclear, which severely restrict their real applications. Here, a 16β steroid-hydroxylase CYP109B4 from Bacillus sonorensis is identified with excellent selectivity and activity. The crystallization and structural analysis of CYP109B4 reveal potential three "hotspot" residues (V84, V292, and S387) responsible for selectivity control. Then, guided by the sequence−function relationships revealed from the mutability landscape construction on the three residues, focused rational iterative site-specific mutagenesis (FRISM) and limited iterative saturation mutagenesis were performed, which provide variant B4-M7 (L240V/S387F/V84L/V292S/I291T/M290F/F294I) with completely switched regioselectivity from 16β to 15β. The subsequent computational analysis uncovers insights into the substrate binding modes in CYP109B4 and its variants, which further confirms the critical role of the "hotspot" residues for selectivity control. Finally, the generality of conserved-"hotspots"mediated selectivity control is demonstrated by performing scaffold sampling between a panel of CYP109B members. Overall, in addition to the present chemical results, our study provides guidance in rationally designing more excellent P450 biocatalysts for potential practical (industrial) applications.
Class I P450 monooxygenase from Rhodococcus coprophilus TC-2, termed P450tol, is the only naturally evolved toluene hydroxylating enzyme known to hydroxylate toluene to produce benzyl alcohol. To investigate its mechanism of action, we solved the unique crystal structures of P450tol and its complex with the substrate. The complex structure indicates that P450tol restricts the toluene binding position with several hydrophobic residues, such that the hydroxylation could take place precisely on the benzylic site. Notably, we found additional space in the toluene-binding pocket and thus examined P450tol activity toward larger substrates. As a result, several halogenated toluenes can also be hydroxylated by P450tol on the benzylic site. We also conducted site saturation mutagenesis (SSM) to enable subterminal or benzylic hydroxylation of propylbenzene. The resulting enantiopure alcohols are essential intermediates for the synthesis of important pharmaceuticals. To facilitate further applications, we fused P450tol and reductase domain derived from self-sufficient P450s. The chimeric enzymes containing the CYP116B46 reductase domain from thermophilic Tepidiphilus thermophiles (P450tol-CYP116B46) exhibit higher thermostability and catalytic activity than the one containing RhFRED reductase domain from mesophilic Rhodococcus sp. strain NCIMB 9784. In conclusion, we manifested the origin of regioselectivity of P450tol-catalyzed benzylic hydroxylation and explored the versatility in substrate utilization of P450tol. Furthermore, the self-sufficient chimeric enzyme with high catalytic activity and stability was generated. We are convinced that these results highlight the great potentials of P450tol in biotechnological and pharmaceutical applications.
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