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Crystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2
Andrew Stewart1, Philip Dershwitz1, Charles Stewart2
1Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011-3260, USA.
Abstract:
Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.
Insights
Methanobactins (MBs), essential for copper acquisition by methanotrophs, undergo complex modifications. This study reveals the structure and function of MbnF, a flavin-dependent enzyme crucial for MB precursor peptide processing.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) vital for copper acquisition in methanotrophs.
- The biosynthesis of MBs involves complex post-translational modifications, including the formation of heterocyclic rings from specific dipeptides, but key enzymes remain uncharacterized.
- MbnF is a putative flavin-dependent enzyme implicated in MB biosynthesis, particularly for pyrazinedione or imidazolone ring formation.
Purpose of the Study:
- To elucidate the function of MbnF, a protein involved in methanobactin biosynthesis.
- To determine the structural characteristics of MbnF and its potential role in precursor peptide modification.
- To investigate the interaction of MbnF with the methanobactin precursor peptide (MbnA).
Main Methods:
- Recombinant production of MbnF in Escherichia coli.
- X-ray crystallography to determine the 3D structure of MbnF.
- Preliminary functional assays including cofactor preference and substrate binding studies.
- Molecular dynamics simulations to analyze MbnF-MbnA interactions.
Main Results:
- The X-ray crystal structure of MbnF was resolved to 2.6 Å resolution, revealing it as a type A flavin-dependent monooxygenase (FMO).
- MbnF preferentially utilizes NADPH over NADH, consistent with NAD(P)H-dependent flavin reduction in FMOs.
- MbnF binds the MB precursor peptide (MbnA), leading to the processing of the leader peptide and the C-terminal region, with molecular dynamics simulations showing a channel accommodating the MbnA core fragment.
Conclusions:
- MbnF is a type A FMO that binds and processes the MB precursor peptide MbnA.
- The structural and functional data suggest MbnF plays a role in the initial steps of methanobactin maturation.
- Further characterization of MbnF provides insights into the complex biosynthetic pathways of methanobactins.
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