Dataset Information

The novel metallo-?-lactamase PNGM-1 from a deep-sea sediment metagenome: crystallization and X-ray crystallographic analysis.

ABSTRACT: Metallo-?-lactamases (MBLs) are present in major Gram-negative pathogens and environmental species, and pose great health risks because of their ability to hydrolyze the ?-lactam rings of antibiotics such as carbapenems. PNGM-1 was the first reported case of a subclass B3 MBL protein that was identified from a metagenomic library from deep-sea sediments that predate the antibiotic era. In this study, PNGM-1 was overexpressed, purified and crystallized. Crystals of native and selenomethionine-substituted PNGM-1 diffracted to 2.10 and 2.30?Å resolution, respectively. Both the native and the selenomethionine-labelled PNGM-1 crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 122, b = 83, c = 163?Å, ? = 110°. Matthews coefficient (VM) calculations suggested the presence of 6-10 molecules in the asymmetric unit, corresponding to a solvent content of ?31-58%. Structure determination is currently in progress.

SUBMITTER: Park KS

PROVIDER: S-EPMC6168767 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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The novel metallo-β-lactamase PNGM-1 from a deep-sea sediment metagenome: crystallization and X-ray crystallographic analysis.

Park Kwang Seung KS Hong Myoung Ki MK Jeon Jin Wan JW Kim Ji Hwan JH Jeon Jeong Ho JH Lee Jung Hun JH Kim Tae Yeong TY Karim Asad Mustafa AM Malik Sumera Kausar SK Kang Lin Woo LW Lee Sang Hee SH

Acta crystallographica. Section F, Structural biology communications 20180919 Pt 10

Metallo-β-lactamases (MBLs) are present in major Gram-negative pathogens and environmental species, and pose great health risks because of their ability to hydrolyze the β-lactam rings of antibiotics such as carbapenems. PNGM-1 was the first reported case of a subclass B3 MBL protein that was identified from a metagenomic library from deep-sea sediments that predate the antibiotic era. In this study, PNGM-1 was overexpressed, purified and crystallized. Crystals of native and selenomethionine-sub ...[more]

PMID: 30279316

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Project description:BACKGROUND:The deep-sea environment harbors a vast pool of novel enzymes. Owing to the limitations of cultivation, cultivation-independent has become an effective method for mining novel enzymes from the environment. Based on a deep-sea sediment metagenomics library, lipolytic-positive clones were obtained by activity-based screening methods. RESULTS:Two novel esterases, DMWf18-543 and DMWf18-558, were obtained from a deep-sea metagenomic library through activity-based screening and high-throughput sequencing methods. These esterases shared 80.7% amino acid identity with each other and were determined to be new members of bacterial lipolytic enzyme family IV. The two enzymes showed the highest activities toward p-nitrophenyl (p-NP) butyrate at pH 7.0 and 35-40 °C and were found to be resistant to some metal ions (Ba2+, Mg2+, and Sr2+) and detergents (Triton X-100, Tween 20, and Tween 80). DMWf18-543 and DMWf18-558 exhibited distinct substrate specificities and preferences. DMWf18-543 showed a catalytic range for substrates of C2-C8, whereas DMWf18-558 presented a wider range of C2-C14. Additionally, DMWf18-543 preferred p-NP butyrate, whereas DMWf18-558 preferred both p-NP butyrate and p-NP hexanoate. To investigate the mechanism underlying the phenotypic differences between the esterases, their three-dimensional structures were compared by using homology modeling. The results suggested that residue Leu199 of DMWf18-543 shortens and blocks the substrate-binding pocket. This hypothesis was confirmed by the finding that the DMWf18-558-A199L mutant showed a similar substrate specificity profile to that of DMWf18-543. CONCLUSIONS:This study characterized two novel homologous esterases obtained from a deep-sea sediment metagenomic library. The structural modeling and mutagenesis analysis provided insight into the determinants of their substrate specificity and preference. The characterization and mechanistic analyses of these two novel enzymes should provide a basis for further exploration of their potential biotechnological applications.

Dataset Information

The novel metallo-?-lactamase PNGM-1 from a deep-sea sediment metagenome: crystallization and X-ray crystallographic analysis.

Publications

The novel metallo-β-lactamase PNGM-1 from a deep-sea sediment metagenome: crystallization and X-ray crystallographic analysis.

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