Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 64
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Org Chem ; 85(3): 1537-1547, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31774678

RESUMO

The glycopeptide antibiotics (GPAs) serve as an important example of the interplay of two powerful enzymatic classes in secondary metabolism: the coupling of nonribosomal peptide synthesis with oxidative aromatic cross-linking performed by cytochrome P450 enzymes. This interplay is responsible for the generation of the highly cross-linked peptide aglycone at the core of this compound class that is required for antibiotic activity and, as such, serves as an important point for the exploration of chemoenzymatic routes to understand the selectivity and mechanism of this complex cascade. Here, we demonstrate the effective reconstitution of enzymatic tetracyclization of synthetic teicoplanin-derived heptapeptides and furthermore discern the importance of the OxyE enzyme in maintaining effective cyclization of such peptides bearing 3,5-dihydroxyphenylglycine residues at position 3 in their structures. These results demonstrate the value of chemically synthesized probes for the elucidation of the enzyme mechanism underpinning the complex process of GPA cyclization and furthermore show the utility of the technique for probing the cyclization of non-natural GPA peptides by these powerful biosynthetic enzymes.

2.
J Biol Chem ; 294(52): 20185-20195, 2019 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-31740583

RESUMO

Expression of human leukocyte antigen (HLA)-B27 is strongly associated with predisposition toward ankylosing spondylitis (AS) and other spondyloarthropathies. However, the exact involvement of HLA-B27 in disease initiation and progression remains unclear. The homodimer theory, which proposes that HLA-B27 heavy chains aberrantly form homodimers, is a central hypothesis that attempts to explain the role of HLA-B27 in disease pathogenesis. Here, we examined the ability of the eight most prevalent HLA-B27 allotypes (HLA-B*27:02 to HLA-B*27:09) to form homodimers. We observed that HLA-B*27:03, a disease-associated HLA-B27 subtype, showed a significantly reduced ability to form homodimers compared with all other allotypes, including the non-disease-associated/protective allotypes HLA-B*27:06 and HLA-B*27:09. We used X-ray crystallography and site-directed mutagenesis to unravel the molecular and structural mechanisms in HLA-B*27:03 that are responsible for its compromised ability to form homodimers. We show that polymorphism at position 59, which differentiates HLA-B*27:03 from all other allotypes, is responsible for its compromised ability to form homodimers. Indeed, histidine 59 in HLA-B*27:03 leads to a series of local conformational changes that act in concert to reduce the accessibility of the nearby cysteine 67, an essential amino acid residue for the formation of HLA-B27 homodimers. Considered together, the ability of both protective and disease-associated HLA-B27 allotypes to form homodimers and the failure of HLA-B*27:03 to form homodimers challenge the role of HLA-B27 homodimers in AS pathoetiology. Rather, this work implicates other features, such as peptide binding and antigen presentation, as pivotal mechanisms for disease pathogenesis.

3.
ACS Chem Biol ; 14(12): 2932-2941, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31774267

RESUMO

ß-Hydroxylation plays an important role in the nonribosomal peptide biosynthesis of many important natural products, including bleomycin, chloramphenicol, and the glycopeptide antibiotics (GPAs). Various oxidative enzymes have been implicated in such a process, with the mechanism of incorporation varying from installation of hydroxyl groups in amino acid precursors prior to adenylation to direct amino acid oxidation during peptide assembly. In this work, we demonstrate the in vitro utility and scope of the unusual nonheme diiron monooxygenase CmlA from chloramphenicol biosynthesis for the ß-hydroxylation of a diverse range of carrier protein bound substrates by adapting this enzyme as a non-native trans-acting enzyme within NRPS-mediated GPA biosynthesis. The results from our study show that CmlA has a broad substrate specificity for modified phenylalanine/tyrosine residues as substrates and can be used in a practical strategy to functionally cross complement compatible NRPS biosynthesis pathways in vitro.

4.
J Biol Chem ; 294(49): 18769-18783, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31672921

RESUMO

Since the discovery of vancomycin in the 1950s, the glycopeptide antibiotics (GPAs) have been of great interest to the scientific community. These nonribosomally biosynthesized peptides are highly cross-linked, often glycosylated, and inhibit bacterial cell wall assembly by interfering with peptidoglycan synthesis. Interest in glycopeptide antibiotics covers many scientific disciplines, due to their challenging total syntheses, complex biosynthesis pathways, mechanism of action, and high potency. After intense efforts, early enthusiasm has given way to a recognition of the challenges in chemically synthesizing GPAs and of the effort needed to study and modify GPA-producing strains to prepare new GPAs to address the increasing threat of microbial antibiotic resistance. Although the preparation of GPAs, either by modifying the pendant groups such as saccharides or by functionalizing the N- or C-terminal moieties, is readily achievable, the peptide core of these molecules-the GPA aglycone-remains highly challenging to modify. This review aims to present a summary of the results of GPA modification obtained with the three major approaches developed to date: in vivo strain manipulation, total chemical synthesis, and chemoenzymatic synthesis methods.

5.
J Biol Chem ; 294(50): 18980-18991, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31624148

RESUMO

To persist when nutrient sources are limited, aerobic soil bacteria metabolize atmospheric hydrogen (H2). This process is the primary sink in the global H2 cycle and supports the productivity of microbes in oligotrophic environments. H2-metabolizing bacteria possess [NiFe] hydrogenases that oxidize H2 to subatmospheric concentrations. The soil saprophyte Mycobacterium smegmatis has two such [NiFe] hydrogenases, designated Huc and Hhy, that belong to different phylogenetic subgroups. Both Huc and Hhy are oxygen-tolerant, oxidize H2 to subatmospheric concentrations, and enhance bacterial survival during hypoxia and carbon limitation. Why does M. smegmatis require two hydrogenases with a seemingly similar function? In this work, we resolved this question by showing that Huc and Hhy are differentially expressed, localized, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phases, supporting energy conservation during mixotrophic growth and transition into dormancy. In contrast, Hhy is most active during long-term persistence, providing energy for maintenance processes following carbon exhaustion. We also show that Huc and Hhy are obligately linked to the aerobic respiratory chain via the menaquinone pool and are differentially affected by respiratory uncouplers. Consistently, these two enzymes interacted differentially with the respiratory terminal oxidases. Huc exclusively donated electrons to, and possibly physically associated with, the proton-pumping cytochrome bcc-aa 3 supercomplex. In contrast the more promiscuous Hhy also provided electrons to the cytochrome bd oxidase complex. These results indicate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.

6.
Org Lett ; 21(21): 8635-8640, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31603691

RESUMO

Natural products are the greatest source of antimicrobial agents, although their structural complexity often renders synthetic production and diversification of key classes impractical. One pertinent example is the glycopeptide antibiotics (GPAs), which are highly challenging to synthesize due to their heavily cross-linked structures. Here, we report an optimized method that generates >75% tricyclic peptides from synthetic precursors in order to explore the acceptance of novel GPA precursor peptides by these key existent biosynthetic enzymes.

7.
Nat Commun ; 10(1): 2613, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197182

RESUMO

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.


Assuntos
Actinobacteria/metabolismo , Antibacterianos/metabolismo , Vias Biossintéticas/genética , Glicopeptídeos/biossíntese , Peptídeos/metabolismo , Actinobacteria/genética , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Ciclização/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/química , Família Multigênica , Peptídeos/química
8.
Chem Sci ; 10(1): 118-133, 2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30713624

RESUMO

Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many of the products of these assembly lines, understanding the activity and selectivity of non-ribosomal peptide synthetase (NRPS) machineries is an essential step towards the redesign of such machineries to produce new bioactive peptides. Whilst the selectivity of the adenylation domains responsible for amino acid activation during NRPS synthesis has been widely studied, the selectivity of the essential peptide bond forming domains - known as condensation domains - is not well understood. Here, we present the results of a combination of in vitro and in vivo investigations into the final condensation domain from the NRPS machinery that produces the glycopeptide antibiotics (GPAs). Our results show that this condensation domain is tolerant for a range of peptide substrates and even those with unnatural stereochemistry of the peptide C-terminus, which is in contrast to the widely ascribed role of these domains as a stereochemical gatekeeper during NRPS synthesis. Furthermore, we show that this condensation domain has a significant preference for linear peptide substrates over crosslinked peptides, which indicates that the GPA crosslinking cascade targets the heptapeptide bound to the final module of the NRPS machinery and reinforces the role of the unique GPA X-domain in this process. Finally, we demonstrate that the peptide bond forming activity of this condensation domain is coupled to the rate of amino acid activation performed by the subsequent adenylation domain. This is a significant result with implications for NRPS redesign, as it indicates that the rate of amino acid activation of modified adenylation domains must be maintained to prevent unwanted peptide hydrolysis from the NRPS due to a loss of the productive coupling of amino acid selection and peptide bond formation. Taken together, our results indicate that assessing condensation domain activity is a vital step in not only understanding the biosynthetic logic and timing of NRPS-mediated peptide assembly, but also the rules which redesign efforts must obey in order to successfully produce functional, modified NRPS assembly lines.

9.
Methods Enzymol ; 617: 113-154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30784400

RESUMO

Nonribosomal peptide biosynthesis is a complex enzymatic assembly responsible for producing a great diversity of bioactive peptide natural products. Due to the recurring arrangement of catalytic domains within these machineries, great interest has been shown in reengineering these pathways to produce novel, designer peptide products. However, in order to realize such ambitions, it is first necessary to develop a comprehensive understanding of the selectivity, mechanisms, and structure of these complex enzymes, which in turn requires significant in vitro experiments. Within nonribosomal biosynthesis, some modifications are performed by enzymatic domains that are not linked to the main nonribosomal peptide synthetase but rather act in trans: these systems offer great potential for redesign, but in turn require detailed study. In this chapter, we present an overview of in vitro experiments that can be used to characterize examples of such trans-interacting enzymes from nonribosomal peptide biosynthesis: Cytochrome P450 monooxygenases and flavin-dependent halogenases.


Assuntos
Aminoácidos/metabolismo , Bactérias/enzimologia , Fungos/enzimologia , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeos/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Modelos Moleculares , Peptídeo Sintases/metabolismo , Especificidade por Substrato
10.
Proc Natl Acad Sci U S A ; 116(8): 2913-2918, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30705105

RESUMO

The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with ß-alanine. Ebony is a rare example of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine-N-acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic example of an alternative type of NRPS condensation domain; they also illustrate how the catalytic components of such assembly lines are significantly more diverse than a minimal set of conserved functional domains.


Assuntos
Arilalquilamina N-Acetiltransferase/química , Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Peptídeo Sintases/química , Animais , Domínio Catalítico , Cristalografia por Raios X , Drosophila melanogaster/química , Domínios Proteicos , Dobramento de Proteína , Estrutura Terciária de Proteína
12.
Nat Prod Rep ; 35(11): 1120-1139, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30207358

RESUMO

Covering: up to July 2018 Non-ribosomal peptide synthetase (NRPS) machineries are complex, multi-domain proteins that are responsible for the biosynthesis of many important, peptide-derived compounds. By decoupling peptide synthesis from the ribosome, NRPS assembly lines are able to access a significant pool of amino acid monomers for peptide synthesis. This is combined with a modular protein architecture that allows for great variation in stereochemistry, peptide length, cyclisation state and further modifications. The architecture of NRPS assembly lines relies upon a repetitive set of catalytic domains, which are organised into modules responsible for amino acid incorporation. Central to NRPS-mediated biosynthesis is the carrier protein (CP) domain, to which all intermediates following initial monomer activation are bound during peptide synthesis up until the final handover to the thioesterase domain that cleaves the mature peptide from the NRPS. This mechanism makes understanding the protein-protein interactions that occur between different NRPS domains during peptide biosynthesis of crucial importance to understanding overall NRPS function. This endeavour is also highly challenging due to the inherent flexibility and dynamics of NRPS systems. In this review, we present the current state of understanding of the protein-protein interactions that govern NRPS-mediated biosynthesis, with a focus on insights gained from structural studies relating to CP domain interactions within these impressive peptide assembly lines.


Assuntos
Biossíntese de Peptídeos Independentes de Ácido Nucleico/fisiologia , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Mapas de Interação de Proteínas/fisiologia , Aminoácidos/metabolismo , Ciclização , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Domínios Proteicos , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo
13.
J Org Chem ; 83(13): 7206-7214, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29708747

RESUMO

Natural products such as the glycopeptide antibiotics (GPAs, including vancomycin and teicoplanin) are of great pharmaceutical importance due to their use against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus. GPAs are assembled in a complex process based on nonribosomal peptide synthesis and late-stage, multistep cross-linking of the linear heptapeptide performed by cytochrome P450 monooxygenases. These P450 enzymes demonstrate varying degrees of substrate selectivity toward the linear peptide precursor, with limited information available about their tolerance regarding modifications to amino acid residues within the essential antibiotic core of the GPA. In order to test the acceptance of altered residues by the P450-catalyzed cyclization cascade, we have explored the use of ß-amino acids in both variable and highly conserved positions within GPA peptides. Our results indicate that the incorporation of ß-amino acids at the C-terminus of the peptide leads to a dramatic reduction in the efficiency of peptide cyclization by the P450s during GPA biosynthesis, whereas replacement of residue 3 is well tolerated by the same enzymes. These results show that maintaining the C-terminal 3,5-dihydroxyphenylglycine residue is of key importance to maintain the efficiency of this complex and essential enzymatic cross-linking process.


Assuntos
Aminoácidos/química , Antibacterianos/biossíntese , Glicopeptídeos/biossíntese , Ciclização , Oxirredução
14.
J Inorg Biochem ; 185: 43-51, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29751197

RESUMO

Cytochrome P450 enzymes perform an impressive range of oxidation reactions against diverse substrate scaffolds whilst generally maintaining a conserved tertiary structure and active site chemistry. Within secondary metabolism, P450 enzymes play widespread and important roles in performing crucial modifications of precursor molecules, with one example of the importance of such reactions being found in the biosynthesis of the glycopeptide antibiotics (GPAs). In GPA biosynthesis P450s, known as Oxy enzymes, are key players in the cyclization of the linear GPA peptide precursor, which is a process that is both essential for their antibiotic activity and is the source of the synthetic challenge of these important antibiotics. In this work, we developed chimeric P450 enzymes from GPA biosynthesis based on two homologues from different GPA biosynthesis pathways - vancomycin and teicoplanin - as an approach to explore the divergent catalytic behavior of the two parental homologues. We could generate, crystalize and explore the activity of new hybrid P450 enzymes from GPA biosynthesis and show that the unusual in vitro behavior of the vancomycin OxyB homologue does not stem from the major regions of the P450 active site, and that additional regions in and around the P450 active site must contribute to the unusual properties of this P450 enzyme. Our results further show that it is possible to successfully transplant entire regions of secondary structure between such P450s and retain P450 expression and activity, which opens the door to use such targeted approaches to generate and explore novel biosynthetic P450 enzymes.


Assuntos
Antibacterianos/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/biossíntese , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Catálise , Cristalização , Sistema Enzimático do Citocromo P-450/química , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
15.
Nat Prod Rep ; 35(8): 757-791, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29667657

RESUMO

Covering: 2000 up to 2018 The cytochromes P450 (P450s) are a superfamily of heme-containing monooxygenases that perform diverse catalytic roles in many species, including bacteria. The P450 superfamily is widely known for the hydroxylation of unactivated C-H bonds, but the diversity of reactions that P450s can perform vastly exceeds this undoubtedly impressive chemical transformation. Within bacteria, P450s play important roles in many biosynthetic and biodegradative processes that span a wide range of secondary metabolite pathways and present diverse chemical transformations. In this review, we aim to provide an overview of the range of chemical transformations that P450 enzymes can catalyse within bacterial secondary metabolism, with the intention to provide an important resource to aid in understanding of the potential roles of P450 enzymes within newly identified bacterial biosynthetic pathways.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Alcaloides/metabolismo , Proteínas de Bactérias/química , Carotenoides/metabolismo , Ácidos Graxos/metabolismo , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Policetídeos/metabolismo , Metabolismo Secundário , Esteroides/metabolismo , Terpenos/metabolismo
16.
Chem Commun (Camb) ; 54(17): 2146-2149, 2018 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-29423498

RESUMO

Non-ribosomal peptides contain an array of amino acid building blocks that can present challenges for the synthesis of important intermediates. Here, we report the synthesis of glycopeptide antibiotic (GPA) thioester peptides that retains the crucial stereochemical purity of the terminal phenylglycine residue, which we show is essential for the enzymatic GPA cyclisation cascade.


Assuntos
Antibacterianos/síntese química , Glicina/análogos & derivados , Glicopeptídeos/síntese química , Antibacterianos/química , Antibacterianos/metabolismo , Vias Biossintéticas , Técnicas de Química Sintética/métodos , Ciclização , Esterificação , Glicina/síntese química , Glicina/metabolismo , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Estereoisomerismo , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo
17.
ACS Chem Biol ; 13(1): 110-120, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29192758

RESUMO

The biosynthesis of the glycopeptide antibiotics (GPAs)-which include teicoplanin and vancomycin-is a complex enzymatic process relying on the interplay of nonribosomal peptide synthesis and a cytochrome P450-mediated cyclization cascade. This unique cyclization cascade generates the highly cross-linked state of these nonribosomal peptides, which is crucial for their antimicrobial activity. Given that these essential oxidative transformations occur while the peptide remains bound to the terminal module of the nonribosomal peptide synthetase (NRPS) machinery, it is important to assess the selectivity of the terminal thioesterase (TE) domain and how this domain contributes to the maintenance of an efficient biosynthetic pathway while at the same time ensuring GPA maturation is completed. In this study, we report the in vitro characterization of the thioesterase domain from teicoplanin biosynthesis, the first GPA thioesterase to be characterized. Our results show that the activity of this TE domain relies on the presence of an unusual extended N-terminal linker region that appears to be unique to the NRPS machineries found in GPA biosynthesis. In addition, we show that the activity of this domain against carrier protein bound substrates is dramatically enhanced for mature GPA aglycones as opposed to linear peptides and partially cyclized intermediates. These results demonstrate how the interplay between NRPS and P450s during late stage GPA biosynthesis is not only maintained but also leads to the efficient production of mature GPA aglycones. Thus, GPA TE domains represent another impressive example of the ability of TE domains to act as logic gates during NRPS biosynthesis, ensuring that essential late-stage peptide modifications are completed before catalyzing the release of the mature, bioactive peptide product.


Assuntos
Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Teicoplanina/biossíntese , Tioléster Hidrolases/química , Peptídeo Sintases/genética , Peptídeos/química , Peptídeos/metabolismo , Domínios Proteicos , Especificidade por Substrato , Tioléster Hidrolases/metabolismo
18.
Biochem Cell Biol ; 96(3): 372-379, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29172027

RESUMO

Non-ribosomal peptide synthetase (NRPS) machineries produce many medically relevant peptides that cannot be easily accessed by chemical synthesis. Thus, understanding NRPS mechanism is of crucial importance to allow efficient redesign of these machineries to produce new compounds. During NRPS-mediated synthesis, substrates are covalently attached to peptidyl carrier proteins (PCPs), and studies of NRPSs are impeded by difficulties in producing PCPs loaded with substrates. Different approaches to load substrates onto PCP domains have been described, but all suffer from difficulties in either the complexity of chemical synthesis or low enzymatic efficiency. Here, we describe an enhanced chemoenzymatic loading method that combines 2 approaches into a single, highly efficient one-pot loading reaction. First, d-pantetheine and ATP are converted into dephospho-coenzyme A via the actions of 2 enzymes from coenzyme A (CoA) biosynthesis. Next, phosphoadenylates are dephosphorylated using alkaline phosphatase to allow linker attachment to PCP domain by Sfp mutant R4-4, which is inhibited by phosphoadenylates. This route does not depend on activity of the commonly problematic dephospho-CoA kinase and, therefore, offers an improved method for substrate loading onto PCP domains.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Peptídeo Sintases/metabolismo , Domínios Proteicos/fisiologia , Sequência de Aminoácidos , Coenzima A/metabolismo , Escherichia coli/metabolismo , Estrutura Terciária de Proteína/fisiologia , Especificidade por Substrato/fisiologia
19.
PLoS Biol ; 15(11): e2003145, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29091712

RESUMO

How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.


Assuntos
Bioquímica/educação , Currículo , Mapas de Interação de Proteínas , Pesquisa/educação , Ensino , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Laboratórios/normas , Aprendizagem , Oxigenases de Função Mista/metabolismo , Estudantes
20.
Chem Sci ; 8(9): 5992-6004, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28989629

RESUMO

Halogenation plays a significant role in the activity of the glycopeptide antibiotics (GPAs), although up until now the timing and therefore exact substrate involved was unclear. Here, we present results combined from in vivo and in vitro studies that reveal the substrates for the halogenase enzymes from GPA biosynthesis as amino acid residues bound to peptidyl carrier protein (PCP)-domains from the non-ribosomal peptide synthetase machinery: no activity was detected upon either free amino acids or PCP-bound peptides. Furthermore, we show that the selectivity of GPA halogenase enzymes depends upon both the structure of the bound amino acid and the PCP domain, rather than being driven solely via the PCP domain. These studies provide the first detailed understanding of how halogenation is performed during GPA biosynthesis and highlight the importance and versatility of trans-acting enzymes that operate during peptide assembly by non-ribosomal peptide synthetases.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA