Methylerythritol phosphate pathway of isoprenoid biosynthesis.

Isoprenoids are a class of natural products with more than 55,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate and its isomer dimethylallyl diphosphate. Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway [the methylerythritol phosphate (MEP) pathway] and a modified mevalonic acid (MVA) pathway. In this review, we summarize mechanistic insights on the MEP pathway enzymes. Because many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both MVA and MEP pathway-based synthetic biology are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations.

In Vitro Characterization of Kitasetaline Biosynthesis Reveals a Bifunctional P450 Decarboxylase and a Vinyl ß-Carboline Intermediate Susceptible to Nonenzymatic Thiol Addition.

Kitasetaline is one of the very few ß-carbolines isolated from bacteria. It features a unique N-acetylcysteine moiety linked to the ß-carboline core through a thioether bond. While earlier in vivo experiments identified the gene cluster and reported several putative biosynthetic intermediates, how the C-S bond linkage is constructed has remained elusive. Herein, in vitro reconstitution of kitasetaline biosynthesis reveals the involvement of a Pictet-Spenglerase (KslB) and a promiscuous dehydrogenase (KslA) that generate the characteristic ß-carboline ring system. In addition, the P450 enzyme KslC was found to catalyze oxidative decarboxylation of 1-(2-carboxyethyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid to yield the biosynthetic intermediate 1-vinyl-9H-pyrido[3,4-b]indole-3-carboxylic acid. KslC is also capable of catalyzing further oxidation of its product to yield an N-hydroxylated side product. Importantly, the vinyl intermediate was found to undergo nonenzymatic nucleophilic addition by N-acetyl-l-cysteine to generate the C-S bond leading directly to kitasetaline without the involvement of a mycothiolated intermediate proposed in a previous biosynthetic model. Thus, this work not only demonstrates that biosynthesis of ß-carboline compounds is rich in unexpected chemistry but also adds to the growing realization that biological thiolation reactions are often nonenzymatic in nature, relying instead on enzymatic formation of reactive electrophiles.

Biosynthesis of the Unusual Epoxy Isonitrile-Containing Antibiotics Aerocyanidin and Amycomicin.

Aerocyanidin and amycomicin are two antibiotics derived from long-chain acids with a rare epoxy isonitrile moiety, the complexity of which renders the total synthesis of these two natural products rather challenging. How this functionality is biosynthesized has also remained obscure. While the biosynthetic gene clusters for these compounds have been identified, both appear to be deficient in genes encoding enzymes seemingly necessary for the oxidative modifications observed in these antibiotics. Herein, the biosynthetic pathways of aerocyanidin and amycomicin are fully elucidated. They share a conserved pathway to isonitrile intermediates that involves a bifunctional thioesterase and a nonheme iron α-ketoglutarate-dependent enzyme. In both cases, the isonitrile intermediates are then loaded onto an acyl carrier protein (ACP) catalyzed by a ligase. The isonitrile-tethered ACP is subsequently processed by polyketide synthase(s) to undergo chain extension, thereby assembling a long-chain γ-hydroxy isonitrile acid skeleton. The epoxide is installed by the cupin domain-containing protein AecF to conclude the biosynthesis of aerocyanidin. In contrast, three P450 enzymes AmcB, AmcC, and AmcQ are involved in epoxidation and keto formation to finalize the biosynthesis of amycomicin. These results thus explain the sequence of oxidation events that result in the final structures of aerocyanidin and amycomicin as well as the biosynthesis of the key γ-hydroxy epoxy isonitrile functional group.

Complete In Vitro Reconstitution of the Apramycin Biosynthetic Pathway Demonstrates the Unusual Incorporation of a ß-d-Sugar Nucleotide in the Final Glycosylation Step.

Apramycin is a widely used aminoglycoside antibiotic with applications in veterinary medicine. It is composed of a 4-amino-4-deoxy-d-glucose moiety and the pseudodisaccharide aprosamine, which is an adduct of 2-deoxystreptamine and an unusual eight-carbon bicyclic dialdose. Despite its extensive study and relevance to medical practice, the biosynthetic pathway of this complex aminoglycoside nevertheless remains incomplete. Herein, the remaining unknown steps of apramycin biosynthesis are reconstituted in vitro, thereby leading to a comprehensive picture of its biological assembly. In particular, phosphomutase AprJ and nucleotide transferase AprK are found to catalyze the conversion of glucose 6-phosphate to NDP-ß-d-glucose as a critical biosynthetic intermediate. Moreover, the dehydrogenase AprD5 and transaminase AprL are identified as modifying this intermediate via introduction of an amino group at the 4″ position without requiring prior 6″-deoxygenation as is typically encountered in aminosugar biosynthesis. Finally, the glycoside hydrolase family 65 protein AprO is shown to utilize NDP-ß-d-glucose or NDP-4"-amino-4"-deoxy-ß-d-glucose to form the 8',1″-O-glycosidic linkage of saccharocin or apramycin, respectively. As the activated sugar nucleotides in all known natural glycosylation reactions involve either NDP-α-d-hexoses or NDP-ß-l-hexoses, the reported chemistry expands the scope of known biological glycosylation reactions to NDP-ß-d-hexoses, with important implications for the understanding and repurposing of aminoglycoside biosynthesis.

Reconstitution of the Final Steps in the Biosynthesis of Valanimycin Reveals the Origin of Its Characteristic Azoxy Moiety.

Valanimycin is an azoxy-containing natural product isolated from the fermentation broth of Streptomyces viridifaciens MG456-hF10. While the biosynthesis of valanimycin has been partially characterized, how the azoxy group is constructed remains obscure. Herein, the membrane protein VlmO and the putative hydrazine synthetase ForJ from the formycin biosynthetic pathway are demonstrated to catalyze N-N bond formation converting O-(l-seryl)-isobutyl hydroxylamine into N-(isobutylamino)-l-serine. Subsequent installation of the azoxy group is shown to be catalyzed by the non-heme diiron enzyme VlmB in a reaction in which the N-N single bond in the VlmO/ForJ product is oxidized by four electrons to yield the azoxy group. The catalytic cycle of VlmB appears to begin with a resting µ-oxo diferric complex in VlmB, as supported by Mössbauer spectroscopy. This study also identifies N-(isobutylamino)-d-serine as an alternative substrate for VlmB leading to two azoxy regioisomers. The reactions catalyzed by the kinase VlmJ and the lyase VlmK during the final steps of valanimycin biosynthesis are established as well. The biosynthesis of valanimycin was thus fully reconstituted in vitro using the enzymes VlmO/ForJ, VlmB, VlmJ and VlmK. Importantly, the VlmB-catalyzed reaction represents the first example of enzyme-catalyzed azoxy formation and is expected to proceed by an atypical mechanism.

Biosynthetic Origin of the Octose Core and Its Mechanism of Assembly during Apramycin Biosynthesis.

Apramycin is an aminoglycoside antibiotic isolated from Streptoalloteichus tenebrarius and S. hindustanus that has found clinical use in veterinary medicine. The apramycin structure is notable for its atypical eight-carbon bicyclic dialdose (octose) moiety. While the apramycin biosynthetic gene cluster (apr) has been identified and several of the encoded genes functionally characterized, how the octose core itself is assembled has remained elusive. Nevertheless, recent gene deletion studies have hinted at an N-acetyl aminosugar being a key precursor to the octose, and this hypothesis is consistent with the additional feeding experiments described in the present report. Moreover, bioinformatic analysis indicates that AprG may be structurally similar to GlcNAc-2-epimerase and hence recognize GlcNAc or a structurally similar substrate suggesting a potential role in octose formation. AprG with an extended N-terminal sequence was therefore expressed, purified, and assayed in vitro demonstrating that it does indeed catalyze a transaldolation reaction between GlcNAc or GalNAc and 6'-oxo-lividamine to afford 7'-N-acetyldemethylaprosamine with the same 6'-R and 7'-S stereochemistry as those observed in the apramycin product. Biosynthesis of the octose core in apramycin thus proceeds in the [6 + 2] manner with GlcNAc or GalNAc as the two-carbon donor, which has not been previously reported for biological octose formation, as well as novel inverting stereochemistry of the transferred fragment. Consequently, AprG appears to be a new transaldolase that lacks any apparent sequence similarity to the currently known aldolases and catalyzes a transaldolation for which there is no established biological precedent.

Changing Fates of the Substrate Radicals Generated in the Active Sites of the B12-Dependent Radical SAM Enzymes OxsB and AlsB.

OxsB is a B12-dependent radical SAM enzyme that catalyzes the oxidative ring contraction of 2'-deoxyadenosine 5'-phosphate to the dehydrogenated, oxetane containing precursor of oxetanocin A phosphate. AlsB is a homologue of OxsB that participates in a similar reaction during the biosynthesis of albucidin. Herein, OxsB and AlsB are shown to also catalyze radical mediated, stereoselective C2'-methylation of 2'-deoxyadenosine monophosphate. This reaction proceeds with inversion of configuration such that the resulting product also possesses a C2' hydrogen atom available for abstraction. However, in contrast to methylation, subsequent rounds of catalysis result in C-C dehydrogenation of the newly added methyl group to yield a 2'-methylidene followed by radical addition of a 5'-deoxyadenosyl moiety to produce a heterodimer. These observations expand the scope of reactions catalyzed by B12-dependent radical SAM enzymes and emphasize the susceptibility of radical intermediates to bifurcation along different reaction pathways even within the highly organized active site of an enzyme.

S-Adenosylmethionine: more than just a methyl donor.

Covering: from 2000 up to the very early part of 2023S-Adenosyl-L-methionine (SAM) is a naturally occurring trialkyl sulfonium molecule that is typically associated with biological methyltransfer reactions. However, SAM is also known to donate methylene, aminocarboxypropyl, adenosyl and amino moieties during natural product biosynthetic reactions. The reaction scope is further expanded as SAM itself can be modified prior to the group transfer such that a SAM-derived carboxymethyl or aminopropyl moiety can also be transferred. Moreover, the sulfonium cation in SAM has itself been found to be critical for several other enzymatic transformations. Thus, while many SAM-dependent enzymes are characterized by a methyltransferase fold, not all of them are necessarily methyltransferases. Furthermore, other SAM-dependent enzymes do not possess such a structural feature suggesting diversification along different evolutionary lineages. Despite the biological versatility of SAM, it nevertheless parallels the chemistry of sulfonium compounds used in organic synthesis. The question thus becomes how enzymes catalyze distinct transformations via subtle differences in their active sites. This review summarizes recent advances in the discovery of novel SAM utilizing enzymes that rely on Lewis acid/base chemistry as opposed to radical mechanisms of catalysis. The examples are categorized based on the presence of a methyltransferase fold and the role played by SAM within the context of known sulfonium chemistry.

A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis.

Oxetanocin A (OXT-A) is a potent antitumour, 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 both the oxsA and oxsB genes are 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, reveals that OXT-A is derived from a 2'-deoxyadenosine phosphate in an OxsB-catalysed ring contraction reaction initiated by hydrogen 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, a family of enzymes with an estimated 7,000 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.

Effects of acute resistance exercise with different loads on appetite, appetite hormones and autonomic nervous system responses in healthy young men.

Although the effect of continuous aerobic exercise on the appetite has been widely explored, the influence of resistance exercise (RE) with different variables, including training loads, training volume, and inter-set rest, on appetite responses requires further investigation. This study examined the importance of training load in RE-induced appetite regulation, with the total training volume and inter-set rest equalized. In total, 11 healthy young men (age = 23 ± 2 years, body mass index = 22 ± 2 kg/m2) were included. Participants completed 3 trials, namely moderate-load RE (MOD; 4 sets of 8 repetitions at 85% 8RM), low-load RE (LOW; 4 sets of 15 repetitions at 45% 8RM), and a control (CON; no exercise), in a randomized, crossover design. Subjective appetite ratings; concentrations of ghrelin, peptide YY (PYY), and lactate; and the autonomic nervous system activity were evaluated before exercise and 1 h after exercise. The hunger and predicted food consumption ratings, and ghrelin concentrations immediately after exercise were significantly lower in the MOD and LOW trials (p < 0.05 vs. CON). The PYY and lactate concentrations immediately after exercise were significantly higher in the MOD and LOW trials (p < 0.05 vs. CON). Heart rate variability recovery was slower in the MOD trial. These findings suggest that both moderate-load and low-load RE at equal training volumes and inter-set rest induce similar responses on hunger suppression and orexigenic signals, except for the slower recovery of autonomic modulation after moderate-load RE. Our results suggest that when individuals aim to potentiate appetite suppression after a bout of RE, both moderate- and low-load RE could be applied.

Studies of lincosamide formation complete the biosynthetic pathway for lincomycin A.

The structure of lincomycin A consists of the unusual eight-carbon thiosugar core methyllincosamide (MTL) decorated with a pendent N-methylprolinyl moiety. Previous studies on MTL biosynthesis have suggested GDP-ᴅ-erythro-α-ᴅ-gluco-octose and GDP-ᴅ-α-ᴅ-lincosamide as key intermediates in the pathway. However, the enzyme-catalyzed reactions resulting in the conversion of GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide have not yet been elucidated. Herein, a biosynthetic subpathway involving the activities of four enzymes-LmbM, LmbL, CcbZ, and CcbS (the LmbZ and LmbS equivalents in the closely related celesticetin pathway)-is reported. These enzymes catalyze the previously unknown biosynthetic steps including 6-epimerization, 6,8-dehydration, 4-epimerization, and 6-transamination that convert GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide. Identification of these reactions completes the description of the entire lincomycin biosynthetic pathway. This work is significant since it not only resolves the missing link in octose core assembly of a thiosugar-containing natural product but also showcases the sophistication in catalytic logic of enzymes involved in carbohydrate transformations.

Characterization of the coformycin biosynthetic gene cluster in Streptomyces kaniharaensis.

Coformycin and pentostatin are structurally related N-nucleoside inhibitors of adenosine deaminase characterized by an unusual 1,3-diazepine nucleobase. Herein, the cof gene cluster responsible for coformycin biosynthesis is identified. Reconstitution of the coformycin biosynthetic pathway in vitro demonstrates that it overlaps significantly with the early stages of l-histidine biosynthesis. Committed entry into the coformycin pathway takes place via conversion of a shared branch point intermediate to 8-ketocoformycin-[Formula: see text]-monophosphate catalyzed by CofB, which is a homolog of succinylaminoimidazolecarboxamide ribotide (SAICAR) synthetase. This reaction appears to proceed via a Dieckmann cyclization and a retro-aldol elimination, releasing ammonia and D-erythronate-4-phosphate as coproducts. Completion of coformycin biosynthesis involves reduction and dephosphorylation of the CofB product, with the former reaction being catalyzed by the NADPH-dependent dehydrogenase CofA. CofB also shows activation by adenosine triphosphate (ATP) despite the reaction requiring neither a phosphorylated nor an adenylated intermediate. This may serve to help regulate metabolic partitioning between the l-histidine and coformycin pathways.

Identification of the Early Steps in Herbicidin Biosynthesis Reveals an Atypical Mechanism of C-Glycosylation.

Herbicidins are adenosine-derived nucleoside antibiotics with an unusual tricyclic core structure. Deletion of the genes responsible for formation of the tricyclic skeleton in Streptomyces sp. L-9-10 reveals the in vivo importance of Her4, Her5, and Her6 in the early stages of herbicidin biosynthesis. In vitro characterization of Her4 and Her5 demonstrates their involvement in an initial, two-stage C-C coupling reaction that results in net C5'-glycosylation of ADP/ATP by UDP/TDP-glucuronic acid. Biochemical analyses and intermediate trapping experiments imply a noncanonical mechanism of C-glycosylation reminiscent of NAD-dependent S-adenosylhomocysteine (SAH)-hydrolase catalysis. Structural characterization of the isolated metabolites suggests possible reactions catalyzed by Her6 and Her7. An overall herbicidin biosynthetic pathway is proposed based on these observations.

Characterization of the Oxazinomycin Biosynthetic Pathway Revealing the Key Role of a Nonheme Iron-Dependent Mono-oxygenase.

Oxazinomycin is a C-nucleoside natural product with antibacterial and antitumor activities. In addition to the characteristic C-glycosidic linkage shared with other C-nucleosides, oxazinomycin also features a structurally unusual 1,3-oxazine moiety, the biosynthesis of which had previously been unknown. Herein, complete in vitro reconstitution of the oxazinomycin biosynthetic pathway is described. Construction of the C-glycosidic bond between ribose 5-phosphate and an oxygen-labile pyridine heterocycle is catalyzed by the C-glycosidase OzmB and involves formation of an enzyme-substrate Schiff base intermediate. The DUF4243 family protein OzmD is shown to catalyze oxygen insertion and rearrangement of the pyridine C-nucleoside intermediate to generate the 1,3-oxazine moiety along with the elimination of cyanide. Spectroscopic analysis and mutagenesis studies indicate that OzmD is a novel nonheme iron-dependent enzyme in which the catalytic iron center is likely coordinated by four histidine residues. These results provide the first example of 1,3-oxazine biosynthesis catalyzed by an unprecedented iron-dependent mono-oxygenase.

Dioxane Bridge Formation during the Biosynthesis of Spectinomycin Involves a Twitch Radical S-Adenosyl Methionine Dehydrogenase That May Have Evolved from an Epimerase.

Spectinomycin is a dioxane-bridged, tricyclic aminoglycoside produced by Streptomyces spectabilis ATCC 27741. While the spe biosynthetic gene cluster for spectinomycin has been reported, the chemistry underlying construction of the dioxane ring is unknown. The twitch radical SAM enzyme SpeY from the spe cluster is shown here to catalyze dehydrogenation of the C2' alcohol of (2'R,3'S)-tetrahydrospectinomycin to yield (3'S)-dihydrospectinomycin as a likely biosynthetic intermediate. This reaction is radical-mediated and initiated via H atom abstraction from C2' of the substrate by the 5'-deoxyadenosyl radical equivalent generated upon reductive cleavage of SAM. Crystallographic analysis of the ternary Michaelis complex places serine-183 adjacent to C2' of the bound substrate opposite C5' of SAM. Mutation of this residue to cysteine converts SpeY to the corresponding C2' epimerase mirroring the opposite phenomenon observed in the homologous twitch radical SAM epimerase HygY from the hygromycin B biosynthetic pathway. Phylogenetic analysis suggests a relatively recent evolutionary branching of putative twitch radical SAM epimerases bearing homologous cysteine residues to generate the SpeY clade of enzymes.

Radical S-Adenosyl Methionine Enzyme BlsE Catalyzes a Radical-Mediated 1,2-Diol Dehydration during the Biosynthesis of Blasticidin S.

The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4'-keto-3'-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4' α-hydroxyalkyl radical as opposed to 1,2-migration of the C3'-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.

Publisher Correction: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Byproduct formation during the biosynthesis of spinosyn A and evidence for an enzymatic interplay to prevent its formation.

Biosynthesis of spinosyn A in Saccharopolyspora spinosa involves a 1,4-dehydration followed by an intramolecular [4 + 2]-cycloaddition catalyzed by SpnM and SpnF, respectively. The cycloaddition also takes place in the absence of SpnF leading to questions regarding its mechanism of catalysis and biosynthetic role. Substrate analogs were prepared with an unactivated dienophile or an acyclic structure and found to be unreactive consistent with the importance of these features for cyclization. The SpnM-catalyzed dehydration reaction was also found to yield a byproduct corresponding to the C11 = C12 cis isomer of the SpnF substrate. This byproduct is stable both in the presence and absence of SpnF; however, relative production of the SpnM product and byproduct could be shifted in favor of the former by including SpnF or the dehydrogenase SpnJ in the reaction. This result suggests a potential interplay between the enzymes of spinosyn A biosynthesis that may help to improve the efficiency of the pathway.

Supervised high-load resistance training for improving muscle strength and quality in prediabetic older adults: A pilot randomized controlled trial.

OBJECTIVE: To investigate the effects of high- and low-load supervised, volume-matched resistance training (RT) on body composition, muscle function, and functional capacity in older adults with prediabetes. METHODS: Older adults with prediabetes were recruited and randomly assigned to high-load RT (n = 13), low-load RT (n = 12), or control groups (n = 12). RESULTS: No significant differences were observed in body composition at postintervention. High-load and low-load RT groups exhibited significant improvements in functional tests at postintervention compared with the control group. The high-load RT group exhibited a greater improvement in muscle strength and muscle quality at postintervention compared with the low-load RT group. CONCLUSION: Supervised RT is useful in the prevention of muscle functional loss in older adults with prediabetes. High-load RT is superior for enhancing muscle strength and muscle quality, despite a similar increase in functional capacity.

Two Radical SAM Enzymes Are Necessary and Sufficient for the In Vitro Production of the Oxetane Nucleoside Antiviral Agent Albucidin.

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 S-adenosyl-l-methionine (SAM) enzyme AlsB, which catalyzes a ring contraction, and the radical SAM enzyme AlsA, which catalyzes elimination of a one-carbon 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.