Summary
Oxetanocin-A (OXT-A, 1) is a potent antitumor, antiviral, and
antibacterial compound. Biosynthesis of OXT-A has been linked to a
plasmid-borne, Bacillus megaterium gene cluster that contains
four genes, oxsA, oxsB, oxrA,
and oxrB. Here, we show that the oxsA and
oxsB genes are both required for the production of OXT-A.
Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent
S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an
HD-domain phosphohydrolase, OxsA, revealed that OXT-A is derived from
2′-deoxyadenosine phosphate in an OxsB-catalyzed ring contraction
reaction initiated by H-atom abstraction from C2′. Hence, OxsB
represents the first biochemically characterized non-methylating Cbl-dependent
AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a
Cbl-dependent AdoMet radical enzyme for which there are an estimated 7000
members. Overall, this work provides a framework for understanding the interplay
of AdoMet and Cbl cofactors and expands the catalytic repertoire of
Cbl-dependent AdoMet radical enzymes.
HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of the Bacillus megaterium HD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A. These studies reveal a previously uncharacterized reaction for this family; OxsA catalyzes the conversion of a triphosphorylated compound into a nucleoside, releasing one molecule of inorganic phosphate at a time. Remarkably, this functionality is a result of the OxsA active site, which based on structural and kinetic analyses has been tailored to bind the small, four-membered ring of oxetanocin-A over larger substrates. Furthermore, our OxsA structures show an active site that switches from a dinuclear to a mononuclear metal center as phosphates are eliminated from substrate.X-ray crystallography | phosphohydrolase | metalloenzymes | natural products | nucleosides
Radical SAM enzymes use S-adenosyl-l-methionine as an oxidant to initiate radical-mediated transformations that would otherwise not be possible with Lewis acid/base chemistry alone. These reactions are either redox neutral or oxidative leading to certain expectations regarding the role of SAM as either a reusable cofactor or the ultimate electron acceptor during each turnover. However, these expectations are frequently not realized resulting in fundamental questions regarding the redox handling and movement of electrons associated with these biological catalysts. Herein we provide a focused perspective on several of these questions and associated hypotheses with an emphasis on recently discovered radical SAM enzymes.
Significance
Dual-function RNAs base pair with target messenger RNAs as small regulatory RNAs and encode small protein regulators. However, only a limited number of these dual-function regulators have been identified. In this study, we show that a well-characterized base-pairing small RNA surprisingly also encodes a 15–amino acid protein. The very small protein binds the cyclic adenosine monophosphate receptor protein transcription factor to block activation of some promoters, raising the question of how many other transcription factors are modulated by unidentified small proteins.
Oxetanocin-A
is an antitumor, antiviral, and antibacterial nucleoside.
It is biosynthesized via the oxidative ring contraction of a purine
nucleoside co-opted from primary metabolism. This reaction is catalyzed
by a B12-dependent radical S-adenosyl-l-methionine (SAM) enzyme, OxsB, and a phosphohydrolase, OxsA.
Previous experiments showed that the product of the OxsB/OxsA-catalyzed
reaction is an oxetane aldehyde produced alongside an uncharacterized
byproduct. Experiments reported herein reveal that OxsB/OxsA complex
formation is crucial for the ring contraction reaction and that reduction
of the aldehyde intermediate is catalyzed by a nonspecific dehydrogenase
from the general cellular pool. In addition, the byproduct is identified
as a 1,3-thiazinane adduct between the aldehyde and l-homocysteine.
While homocysteine was never included in the OxsB/OxsA assays, the
data suggest that it can be generated from SAM via S-adenosyl-l-homocysteine (SAH). Further study revealed that
conversion of SAM to SAH is facilitated by OxsB; however, the subsequent
conversion of SAH to homocysteine is due to protein contaminants that
co-purify with OxsA. Nevertheless, the observed demethylation of SAM
to SAH suggests possible methyltransferase activity of OxsB, and substrate
methylation was indeed detected in the OxsB-catalyzed reaction. This
work is significant because it not only completes the description
of the oxetanocin-A biosynthetic pathway but also suggests that OxsB
may be capable of methyltransferase activity.
Lincomycin A is a clinically useful antibiotic isolated from Streptomyces lincolnensis. It contains an unusual methylmercapto-substituted octose, methylthiolincosamide (MTL). While it has been demonstrated that the C8 backbone of MTL moiety is derived from D-fructose 6-phosphate and D-ribose 5-phosphate via a transaldol reaction catalyzed by LmbR, the subsequent enzymatic transformations leading to the MTL moiety remain elusive. Here, we report the identification of GDP-D-erythro-α-D-gluco-octose (GDP-D-α-D-octose) as a key intermediate in the MTL biosynthetic pathway. Our data show that the octose 1,8-bisphosphate intermediate is first converted to octose 1-phosphate by a phosphatase, LmbK. The subsequent conversion of the octose 1-phosphate to GDP-D-α-D-octose is catalyzed by the octose 1-phosphate guanylyltransferase, LmbO. These results provide significant insight into the lincomycin biosynthetic pathway, because the activated octose likely serves as the acceptor for the installation of the C1 sulfur appendage of MTL.
Oxetanocin A and albucidin are two oxetane natural products. While the biosynthesis of oxetanocin A has been described, less is known about albucidin. In this work, the albucidin biosynthetic gene cluster is identified in Streptomyces. Heterologous expression in a nonproducing strain demonstrates that the genes alsA and alsB are necessary and sufficient for albucidin biosynthesis confirming a previous study (Myronovskyi et al. Microorganisms 2020, 8, 237). A two-step construction of albucidin 4'-phosphate from 2'-deoxyadenosine monophosphate (2'-dAMP) is shown to be catalyzed in vitro by the cobalamin dependent radical Sadenosyl-L-methionine (SAM) enzyme AlsB, which catalyzes a ring contraction, and the radical SAM enzyme AlsA, which catalyzes elimination of a onecarbon fragment. Isotope labelling studies show that AlsB catalysis begins with stereospecific H-atom transfer of the C2'-pro-R hydrogen from 2'-dAMP to 5'deoxyadenosine, and that the eliminated one-carbon fragment originates from C3' of 2'-dAMP.
Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here we report these proteins are new toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic full-length proteins. The crystal structure of RpnAS revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.
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