Dataset Information

Crystal structure of chloramphenicol-metabolizing enzyme EstDL136 from a metagenome.

ABSTRACT: Metagenomes often convey novel biological activities and therefore have gained considerable attention for use in biotechnological applications. Recently, metagenome-derived EstDL136 was found to possess chloramphenicol (Cm)-metabolizing features. Sequence analysis showed EstDL136 to be a member of the hormone-sensitive lipase (HSL) family with an Asp-His-Ser catalytic triad and a notable substrate specificity. In this study, we determined the crystal structures of EstDL136 and in a complex with Cm. Consistent with the high sequence similarity, the structure of EstDL136 is homologous to that of the HSL family. The active site of EstDL136 is a relatively shallow pocket that could accommodate Cm as a substrate as opposed to the long acyl chain substrates typical of the HSL family. Mutational analyses further suggested that several residues in the vicinity of the active site play roles in the Cm-binding of EstDL136. These results provide structural and functional insights into a metagenome-derived EstDL136.

SUBMITTER: Kim SH

PROVIDER: S-EPMC6333409 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Crystal structure of chloramphenicol-metabolizing enzyme EstDL136 from a metagenome.

Kim Sang-Hoon SH Kang Pyeoung-Ann PA Han Keetae K Lee Seon-Woo SW Rhee Sangkee S

PloS one 20190115 1

Metagenomes often convey novel biological activities and therefore have gained considerable attention for use in biotechnological applications. Recently, metagenome-derived EstDL136 was found to possess chloramphenicol (Cm)-metabolizing features. Sequence analysis showed EstDL136 to be a member of the hormone-sensitive lipase (HSL) family with an Asp-His-Ser catalytic triad and a notable substrate specificity. In this study, we determined the crystal structures of EstDL136 and in a complex with ...[more]

PMID: 30645605

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Project description:Enzyme function requires that enzyme structures be dynamic. Substrate binding, product release, and transition state stabilization typically involve different enzyme conformers. Furthermore, in multistep enzyme-catalyzed reactions, more than one enzyme conformation may be important for stabilizing different transition states. While X-ray crystallography provides the most detailed structural information of any current methodology, X-ray crystal structures of enzymes capture only those conformations that fit into the crystal lattice, which may or may not be relevant to function. Solution nuclear magnetic resonance (NMR) methods can provide an alternative approach to characterizing enzymes under nonperturbing and controllable conditions, allowing one to identify and localize dynamic processes that are important to function. However, many enzymes are too large for standard approaches to making sequential resonance assignments, a critical first step in analyzing and interpreting the wealth of information inherent in NMR spectra. This Account describes our long-standing NMR-based research into structural and dynamic aspects of function in the cytochrome P450 monooxygenase superfamily. These heme-containing enzymes typically catalyze the oxidation of unactivated C-H and C═C bonds in a multitude of substrates, often with complete regio- and stereospecificity. Over 600 000 genes in GenBank have been assigned to P450s, yet all known P450 structures exhibit a highly conserved and unique fold. This combination of functional and structural conservation with a vast substrate clientele, each substrate having multiple possible sites for oxidation, makes the P450s a unique target for understanding the role of enzyme structure and dynamics in determining a particular substrate-product combination. P450s are large by solution NMR standards, requiring us to develop specialized approaches for making sequential resonance assignments and interpreting the spectral changes that occur as a function of changing conditions (e.g., oxidation and spin state changes, ligand, substrate or effector binding). Solution conformations are characterized by the fitting of residual dipolar couplings (RDCs) measured for sequence-specifically assigned amide N-H correlations to alignment tensors optimized in the course of restrained molecular dynamics (MD) simulations. The conformational ensembles obtained by such RDC-restrained simulations, which we call "soft annealing", are then tested by site-directed mutation and spectroscopic and activity assays for relevance. These efforts have gained us insights into cryptic conformational changes associated with substrate and redox partner binding that were not suspected from crystal structures, but were shown by subsequent work to be relevant to function. Furthermore, it appears that many of these changes can be generalized to P450s besides those that we have characterized, providing guidance for enzyme engineering efforts. While past research was primarily directed at the more tractable prokaryotic P450s, our current efforts are aimed at medically relevant human enzymes, including CYP17A1, CYP2D6, and CYP3A4.

Dataset Information

Crystal structure of chloramphenicol-metabolizing enzyme EstDL136 from a metagenome.

Publications

Crystal structure of chloramphenicol-metabolizing enzyme EstDL136 from a metagenome.

Similar Datasets

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