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1.
ACS Chem Biol ; 16(4): 701-711, 2021 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-33764747

RESUMEN

N-Glycosylation is a fundamental protein modification found in both eukaryotes and archaea. Despite lacking N-glycans, many commensal and pathogenic bacteria have developed mechanisms to degrade these isoforms for a variety of functions, including nutrient acquisition and evasion of the immune system. Although much is known about many of the enzymes responsible for N-glycan degradation, the enzymes involved in cleaving the N-glycan core have only recently been discovered. Thus, some of the structural details have yet to be characterized, and little is known about their full distribution among bacterial strains and specifically within potential Gram-positive polysaccharide utilization loci. Here, we report crystal structures for Family 5, Subfamily 18 (GH5_18) glycoside hydrolases from the gut bacterium Bifidobacterium longum (BlGH5_18) and the soil bacterium Streptomyces cattleya (ScGH5_18), which hydrolyze the core Manß1-4GlcNAc disaccharide. Structures of these enzymes in complex with Manß1-4GlcNAc reveal a more complete picture of the -1 subsite. They also show that a C-terminal active site cap present in BlGH5_18 is absent in ScGH5_18. Although this C-terminal cap is not widely distributed throughout the GH5_18 family, it is important for full enzyme activity. In addition, we show that GH5_18 enzymes are found in Gram-positive polysaccharide utilization loci that share common genes, likely dedicated to importing and degrading N-glycan core structures.

2.
Nat Chem ; 2021 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-33782561

RESUMEN

Natural products with rare functional groups are likely to be constructed by unique biosynthetic enzymes. One such rare functional group is the O-methyl nitronate, which can undergo [3 + 2] cycloaddition reactions with olefins in mild conditions. O-methyl nitronates are found in some natural products; however, how such O-methyl nitronates are assembled biosynthetically is unknown. Here we show that the assembly of the O-methyl nitronate in the natural product enteromycin carboxamide occurs via activation of glycine on a peptidyl carrier protein, followed by reaction with a diiron oxygenase to give a nitronate intermediate and then with a methyltransferase to give an O-methyl nitronate. Guided by the discovery of this pathway, we then identify related cryptic biosynthetic gene cassettes in other bacteria and show that these alternative gene cassettes can, instead, facilitate oxidative denitrification of glycine-derived nitronates. Altogether, our work reveals bifurcating pathways from a central glycine-derived nitronate intermediate in bacteria.

3.
Anaerobe ; : 102320, 2021 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-33460787

RESUMEN

Bifidobacterium longum subsp. infantis ATCC 15697 has emerged as a model for infant gut-associated bifidobacterial strains. Here we present a genetic system for B. longum subsp. infantis ATCC 15697 using its own DNA restriction-modification systems and create a fucose permease deletion mutant lacking the ability to use free fucose as a carbon source.

4.
ACS Chem Biol ; 2020 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-33232109

RESUMEN

Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species-specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their antimalarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the salinipostins and newly identified natural A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed among many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

6.
mBio ; 11(3)2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32546617

RESUMEN

Plant root-associated microbes promote plant growth and elicit induced systemic resistance (ISR) to foliar pathogens. In an attempt to find novel growth-promoting and ISR-inducing strains, we previously identified strains of root-associated Pseudomonas spp. that promote plant growth but unexpectedly elicited induced systemic susceptibility (ISS) rather than ISR to foliar pathogens. Here, we demonstrate that the ISS-inducing phenotype is common among root-associated Pseudomonas spp. Using comparative genomics, we identified a single Pseudomonas fluorescens locus that is unique to ISS strains. We generated a clean deletion of the 11-gene ISS locus and found that it is necessary for the ISS phenotype. Although the functions of the predicted genes in the locus are not apparent based on similarity to genes of known function, the ISS locus is present in diverse bacteria, and a subset of the genes were previously implicated in pathogenesis in animals. Collectively, these data show that a single bacterial locus contributes to modulation of systemic plant immunity.IMPORTANCE Microbiome-associated bacteria can have diverse effects on health of their hosts, yet the genetic and molecular bases of these effects have largely remained elusive. This work demonstrates that a novel bacterial locus can modulate systemic plant immunity. Additionally, this work demonstrates that growth-promoting strains may have unanticipated consequences for plant immunity, and this is critical to consider when the plant microbiome is being engineered for agronomic improvement.

7.
Org Lett ; 22(11): 4053-4057, 2020 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-32283033

RESUMEN

Methods for the focused isolation of low-abundance natural products with specific chemical substructures could expand known bioactive chemical diversity for drug discovery. Here we report the combined use of genome mining and an 15N NMR-based screening method for the targeted isolation of the low-abundance piperazic-acid-containing peptides incarnatapeptins A (1) and B (3). Incarnatapeptin B (3) shows in vitro cytotoxicity to LNCaP prostate cancer cells.

8.
FEBS J ; 287(7): 1403-1428, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32142210

RESUMEN

Pyridoxal 5'-phosphate (PLP) is an organic cofactor employed by ~ 4% of enzymes. The structure of the PLP cofactor allows for the stabilization of carbanions through resonance. A small number of PLP-dependent enzymes employ molecular oxygen as a cosubstrate. Here, we review the biological roles and possible mechanisms of these enzymes, and we observe that these enzymes are found in multiple protein families, suggesting that reaction with oxygen might have emerged de novo in several protein families and thus could be directed to emerge again through laboratory evolution experiments.

9.
Chem Rev ; 120(6): 3161-3209, 2020 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-31869221

RESUMEN

Natural nonproteinogenic amino acids vastly outnumber the well-known 22 proteinogenic amino acids. Such amino acids are generated in specialized metabolic pathways. In these pathways, diverse biosynthetic transformations, ranging from isomerizations to the stereospecific functionalization of C-H bonds, are employed to generate structural diversity. The resulting nonproteinogenic amino acids can be integrated into more complex natural products. Here we review recently discovered biosynthetic routes to freestanding nonproteinogenic α-amino acids, with an emphasis on work reported between 2013 and mid-2019.

10.
Angew Chem Int Ed Engl ; 59(10): 3881-3885, 2020 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-31823464

RESUMEN

The formation of a N-N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N-N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate-containing amino acid l-alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l-alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl-carrier-protein-based mechanism for activation of the precursor l-diaminopropionate, and we also show that nitric oxide can participate in non-enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N-terminal cupin domain and a C-terminal AraC-like DNA-binding domain, is required for alanosine biosynthesis.

11.
Chembiochem ; 21(5): 644-649, 2020 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-31482654

RESUMEN

Pyrazomycin is a rare C-nucleoside antibiotic containing a naturally occurring pyrazole ring, the biosynthetic origin of which has remained obscure for decades. In this study we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRL 3601, revealing that the StrR-family regulator PyrR is the cluster-situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable-isotope feeding experiments provide support for the hypothesis that PyrN is a new nitrogen-nitrogen bond-forming enzyme that catalyzes the linkage of the ϵ-NH2 nitrogen atom of l-N6 -OH-lysine and the α-NH2 nitrogen atom of l-glutamic acid. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes involved in constructing the characteristic pyrazole ring.

12.
Nat Chem Biol ; 15(11): 1043-1048, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31406372

RESUMEN

Microbes produce specialized metabolites to thrive in their natural habitats. However, it is rare that a given specialized metabolite is biosynthesized via pathways with distinct intermediates and enzymes. Here, we show that the core assembly mechanism of the antibiotic indolmycin in marine gram-negative Pseudoalteromonas luteoviolacea is distinct from its counterpart in terrestrial gram-positive Streptomyces species, with a molecule that is a shunt product in the Streptomyces pathway employed as a biosynthetic substrate for a novel metal-independent N-demethylindolmycin synthase in the P. luteoviolacea pathway. To provide insight into this reaction, we solved the 1.5 Å resolution structure in complex with product and identified the active site residues. Guided by our biosynthetic insights, we then engineered the Streptomyces indolmycin producer for titer improvement. This study provides a paradigm for understanding how two unique routes to a microbial specialized metabolite can emerge from convergent biosynthetic transformations.


Asunto(s)
Bacterias/metabolismo , Vías Biosintéticas , Bacterias/genética , Biocatálisis , Familia de Multigenes
13.
J Am Chem Soc ; 141(31): 12258-12267, 2019 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-31298853

RESUMEN

Acyclic imines are unstable in aqueous conditions. For this reason, known imine reductases, which enable the synthesis of chiral amines, mainly intercept stable cyclic imines. Here we report the detailed biochemical and structural characterization of Bsp5, an imino acid reductase from the d-2-hydroxyacid dehydrogenase family that reduces acyclic imino acids produced in situ by a partner oxidase. We determine a 1.6 Å resolution structure of Bsp5 in complex with d-arginine and coenzyme NADPH. Combined with mutagenesis work, our study reveals the minimal structural constraints for its biosynthetic activity. Furthermore, we demonstrate that Bsp5 can intercept more complex products from an alternate oxidase partner, suggesting that this oxidase-imino acid reductase pair could be evolved for biocatalytic conversion of l-amino acids to d-amino acids.


Asunto(s)
Iminoácidos/química , Iminoácidos/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Biocatálisis , Modelos Moleculares , Dominios Proteicos
14.
Angew Chem Int Ed Engl ; 58(34): 11647-11651, 2019 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-31231913

RESUMEN

Nitroimidazoles are one of the most effective ways to treat anaerobic bacterial infections. Synthetic nitroimidazoles are inspired by the structure of azomycin, isolated from Streptomyces eurocidicus in 1953. Despite its foundational role, no biosynthetic gene cluster for azomycin has been found. Guided by bioinformatics, we identified a cryptic biosynthetic gene cluster in Streptomyces cattleya and then carried out in vitro reconstitution to deduce the enzymatic steps in the pathway linking l-arginine to azomycin. The gene cluster we discovered is widely distributed among soil-dwelling actinobacteria and proteobacteria, suggesting that azomycin and related nitroimidazoles may play important ecological roles. Our work sets the stage for development of biocatalytic approaches to generate azomycin and related nitroimidazoles.


Asunto(s)
Antibacterianos/metabolismo , Arginina/metabolismo , Proteínas Bacterianas/genética , Vías Biosintéticas/genética , Familia de Multigenes , Streptomyces/metabolismo , Proteínas Bacterianas/metabolismo , Nitroimidazoles/metabolismo , Streptomyces/genética , Streptomyces/crecimiento & desarrollo
15.
Nat Prod Rep ; 36(12): 1628-1653, 2019 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-30949650

RESUMEN

Covering: up to the end of 2018 Piperazic acid is a cyclic hydrazine and a non-proteinogenic amino acid found in diverse non-ribosomal peptide (NRP) and hybrid NRP-polyketide (PK) structures. Piperazic acid was first identified as a residue in the monamycins in 1959. Since then, the piperazic acid residue has been found in >30 families of natural products, representing >140 compounds. Many of these compounds have potent biological activity, ranging from anti-malarial to anti-apoptotic to anti-bacterial activity, although high toxicity often accompanies this potent biological activity. Recently, we identified a piperazate synthase, responsible for N-N bond formation to give piperazic acid. Here, we review piperazic acid-containing natural products discovered from 1959 to 2018, with an emphasis on the biosynthetic routes to these natural products.


Asunto(s)
Productos Biológicos/química , Productos Biológicos/metabolismo , Piridazinas/química , Productos Biológicos/farmacología , Estructura Molecular , Familia de Multigenes , Péptidos/química , Péptidos/metabolismo , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Piridazinas/metabolismo
16.
J Am Chem Soc ; 141(9): 4026-4033, 2019 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-30763082

RESUMEN

Nitric oxide (NO) has wide-ranging roles in biology, but less is known about its role in building chemical diversity. Here we report a new route to NO from the biosynthetic pathway to the N-nitroso compound streptozocin. We show that the N-nitroso group of streptozocin comes from the biosynthetic reassembly of l-arginine, with the guanidino nitrogens forming a nitrogen-nitrogen bond. To understand this biosynthetic process, we identify the biosynthetic gene cluster of streptozocin and demonstrate that free l-arginine is N-methylated by StzE to give Nω-monomethyl-l-arginine. We show that this product is then oxidized by StzF, a nonheme iron-dependent enzyme unrelated to known nitric oxide synthases, generating a urea compound and NO. Our work implies that formation and capture of NO is the likely route to N-nitroso formation in vivo. Altogether, our work unveils a new enzyme pair for the production of NO from l-arginine and sets the stage for understanding biosynthetic routes to N-nitroso natural products.


Asunto(s)
Arginina/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Compuestos Nitrosos/metabolismo , Proteínas de Hierro no Heme/metabolismo , Arginina/química , Estructura Molecular , Óxido Nítrico/química , Compuestos Nitrosos/química
17.
Nat Prod Rep ; 36(3): 430-457, 2019 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-30183796

RESUMEN

Covering: up to mid-2018 Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and ß- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.


Asunto(s)
Productos Biológicos/metabolismo , Fosfato de Piridoxal/fisiología , Imidazoles , Lactamas/metabolismo , Lipoproteínas/biosíntesis , Macrólidos/metabolismo , Oligopéptidos/biosíntesis , Pactamicina/biosíntesis , Saxitoxina/biosíntesis , Tiazoles/metabolismo , Tionas/metabolismo
19.
ACS Chem Biol ; 13(4): 965-974, 2018 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-29466666

RESUMEN

Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme and nonheme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)-dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts l-arginine to ( S)-4-hydroxy-2-ketoarginine. We determine that the RohP reaction consumes oxygen with stoichiometric release of H2O2. To understand this unusual chemistry, we obtain ∼1.5 Šresolution structures that capture intermediates along the catalytic cycle. Our data suggest that RohP carries out a four-electron oxidation and a stereospecific alkene hydration to give the ( S)-configured product. Together with our earlier studies on an O2, PLP-dependent l-arginine oxidase, our work suggests that there is a shared pathway leading to both oxidized and hydroxylated products from l-arginine.


Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Oxígeno/química , Fosfato de Piridoxal , Aminoácido Oxidorreductasas/metabolismo , Arginina , Catálisis , Cristalografía por Rayos X , Peróxido de Hidrógeno , Hidroxilación , Oxidación-Reducción
20.
Nat Chem Biol ; 13(8): 836-838, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28628093

RESUMEN

Molecules containing a nitrogen-nitrogen (N-N) linkage have a variety of structures and biological activities; however, no enzyme has yet been demonstrated to catalyze N-N bond formation in an organic molecule. Here we report that the heme-dependent enzyme KtzT from Kutzneria sp. 744 catalyzes N-N bond formation in the biosynthesis of piperazate, a building block for nonribosomal peptides.


Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Nitrógeno/metabolismo , Piridazinas/metabolismo , Actinomycetales/enzimología , Oxigenasas de Función Mixta/química , Conformación Molecular , Nitrógeno/química , Piridazinas/química
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