Crystal Structure of Methylornithine Synthase (PylB): Insights into the Pyrrolysine Biosynthesis

Quitterer, F.; List, A.; Eisenreich, Wolfgang; Bacher, Adelbert; Groll, M.

doi:10.1002/anie.201106765

Cited by 50 publications

(74 citation statements)

References 21 publications

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“…S1). In this OPEN conformation, residues in the α4 helix (121-134), a loop following the AdoMet cluster binding loop (28)(29)(30)(31)(32), and a linker region connecting the N-terminal AdoMet domain to the C-terminal auxiliary cluster domain (146-161) are disordered and not included in the model. In addition, electron density for BtrN substrates AdoMet and DOIA, which were included in the crystallization conditions, is not observed in this OPEN structure.…”

Section: Resultsmentioning

confidence: 99%

“…S2 and S3). In addition, the fold contains a basic residue, H117, that interacts with the carboxyl group of AdoMet, as seen in HemN (29), HydE (30), PylB (31), and anSMEcpe (20), and another hydrogen bond common to nearly all AdoMet radical members, between the N6 position of AdoMet and the carbonyl of Y22, the hydrophobic residue of the AdoMet radical cluster binding motif (16) (Fig. S3).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Goldman

Grove²,

Booker

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The 2-deoxy-scyllo-inosamine (DOIA) dehydrogenases are key enzymes in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics. In contrast to most DOIA dehydrogenases, which are NAD-dependent, the DOIA dehydrogenase from Bacillus circulans (BtrN) is an S-adenosyl-L-methionine (AdoMet) radical enzyme. To examine how BtrN employs AdoMet radical chemistry, we have determined its structure with AdoMet and substrate to 1.56 Å resolution. We find a previously undescribed modification to the core AdoMet radical fold: instead of the canonical (β/α) 6 architecture, BtrN displays a (β 5 /α 4 ) motif. We further find that an auxiliary [4Fe-4S] cluster in BtrN, thought to bind substrate, is instead implicated in substrate-radical oxidation. High structural homology in the auxiliary cluster binding region between BtrN, fellow AdoMet radical dehydrogenase anSME, and molybdenum cofactor biosynthetic enzyme MoaA provides support for the establishment of an AdoMet radical structural motif that is likely common to ∼6,400 uncharacterized AdoMet radical enzymes.radical SAM enzyme | iron-sulfur cluster fold | twitch domain D ue to mounting antibiotic resistance, the discovery and/or modification of antibiotic compounds to fight bacterial infection is a vital task of our time (1). Understanding antibiotic biosynthesis is an important first step in being able to engineer a new wave of therapeutic molecules. Aminoglycosides are a class of antibiotics that inhibit protein synthesis by interacting with the 30S subunit of the bacterial ribosome and have had considerable success in the clinical setting (2). Most FDA-approved aminoglycosides, including neomycin, gentamicin, and kanamycin, share a common glucose-6-phosphate-derived 2-deoxystreptamine (DOS) structural core (3) (blue in Scheme 1). Although the majority of aminoglycoside-producing bacteria use similar reaction sequences to biosynthesize this DOS core, including the penultimate two-electron oxidation of 2-deoxy-scyllo-inosamine (DOIA) to amino-dideoxy-scyllo-inosose (amino-DOI) (Scheme 1, A), identification of the enzymes responsible has not always been straightforward. For example, the btrE gene product in the butirosin B producing Bacillus circulans was proposed to be an NADdependent enzyme responsible for the generation of amino-DOI, as in other aminoglycoside pathways (4). This hypothesis proved incorrect upon further sequence and biochemical analysis (5, 6). Instead, Yokoyama et al. found that production of amino-DOI required another enzyme, BtrN, an S-adenosyl-L-methionine (AdoMet, SAM) radical dehydrogenase (5). Here, we report structures of catalytic and noncatalytic forms of BtrN, providing a structural basis for the unique reaction it performs.The AdoMet radical enzyme family catalyzes a diverse array of radical-based reactions, including sulfur insertions, complex chemical transformations and rearrangements, DNA and RNA modifications, and, in the case of BtrN, dehydrogenation (7). BtrN is one of two known members of the dehydrogenase subfamily of ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Goldman

Grove²,

Booker

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…PylB is a TIM barrel (Fig. 3B), with a central cavity containing the catalytic (4Fe-4S) cluster and S-adenosylmethionine (42). The recombinant PylB is catalytically active without the extension of the 21-amino acid residue Pyl peptide (18).…”

Section: Discussionmentioning

confidence: 99%

Reducing the genetic code induces massive rearrangement of the proteome

O’Donoghue

Prat²,

Kucklick

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Expanding the genetic code is an important aim of synthetic biology, but some organisms developed naturally expanded genetic codes long ago over the course of evolution. Less than 1% of all sequenced genomes encode an operon that reassigns the stop codon UAG to pyrrolysine (Pyl), a genetic code variant that results from the biosynthesis of Pyl-tRNA Pyl . To understand the selective advantage of genetically encoding more than 20 amino acids, we constructed a markerless tRNA Pyl deletion strain of Methanosarcina acetivorans (ΔpylT) that cannot decode UAG as Pyl or grow on trimethylamine. Phenotypic defects in the ΔpylT strain were evident in minimal medium containing methanol. Proteomic analyses of wild type (WT) M. acetivorans and ΔpylT cells identified 841 proteins from >7,000 significant peptides detected by MS/MS. Protein production from UAG-containing mRNAs was verified for 19 proteins. Translation of UAG codons was verified by MS/MS for eight proteins, including identification of a Pyl residue in PylB, which catalyzes the first step of Pyl biosynthesis. Deletion of tRNA Pyl globally altered the proteome, leading to >300 differentially abundant proteins. Reduction of the genetic code from 21 to 20 amino acids led to significant down-regulation in translation initiation factors, amino acid metabolism, and methanogenesis from methanol, which was offset by a compensatory (100-fold) up-regulation in dimethyl sulfide metabolic enzymes. The data show how a natural proteome adapts to genetic code reduction and indicate that the selective value of an expanded genetic code is related to carbon source range and metabolic efficiency.evolution | genetic code expansion | methanogenesis | pyrrolysine | tRNA Pyl

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“…The asymmetric unit contains two HydG monomers and the core structure of HydG is a complete triose phosphate isomerase (TIM) barrel, typical of RS enzymes the bind a small second substrate (such as PylB [21], BioB [22] and NosL [11]) (Figure 2a). The RS and auxiliary clusters are bound at either end of the TIM barrel, separated by more than 23 Å (Figure 2b) and, unusually for RS enzymes, HydG includes a C-terminal helical domain (80 amino acid residues) that encloses the auxiliary cluster ( Figure 2a).…”

Section: Structure Of Hydgmentioning

confidence: 99%

Metallocofactor assembly for [FeFe]-hydrogenases

Dinis

Wieckowski

Roach

2016

Current Opinion in Structural Biology

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Hydrogenases are a potential source of environmentally benign bioenergy, using complex cofactors to catalyze the reversible reduction of protons to form hydrogen. AddressesChemistry and the Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK.Corresponding author: Peter L. Roach (plr2@soton.ac.uk). AbstractHydrogenases are a potential source of environmentally benign bioenergy, using complex cofactors to catalyze the reversible reduction of protons to form hydrogen. The most active subclass, the [FeFe]-hydrogenases, is dependent on a metallocofactor, the H cluster, that consists of a two iron subcluster ([2Fe] H ) bridging to a classical cubane cluster ([4Fe-4S] H ). The ligands coordinating to the diiron subcluster include an azadithiolate, three carbon monoxides, and two cyanides. To assemble this complex cofactor, three maturase enzymes, HydG, HydE and HydF are required. The biosynthesis of the diatomic ligands proceeds by an unusual fragmentation mechanism, and structural studies in combination with spectroscopic analysis have started to provide insights into the HydG mediated assembly of a [2Fe] H subcluster precursor.

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Crystal Structure of Methylornithine Synthase (PylB): Insights into the Pyrrolysine Biosynthesis

Cited by 50 publications

References 21 publications

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Reducing the genetic code induces massive rearrangement of the proteome

Metallocofactor assembly for [FeFe]-hydrogenases

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