Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 15 de 15
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Biochemistry ; 62(21): 3105-3115, 2023 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-37890134

RESUMO

MppQ is an enzyme of unknown function from Streptomyces hygroscopicus (ShMppQ) that operates in the biosynthesis of the nonproteinogenic amino acid L-enduracididine (L-End). Since L-End is a component of several peptides showing activity against antibiotic-resistant pathogens, understanding its biosynthetic pathway could facilitate the development of chemoenzymatic routes to novel antibiotics. Herein, we report on the crystal structures of ShMppQ complexed with pyridoxal-5'-phosphate (PLP) and pyridoxamine-5'-phosphate (PMP). ShMppQ is similar to fold-type I PLP-dependent aminotransferases like aspartate aminotransferase. The tertiary structure of ShMppQ is composed of an N-terminal extension, a large domain, and a small domain. The active site is placed at the junction of the large and small domains and includes residues from both protomers of the homodimer. We also report the first functional characterization of MppQ, which we incubated with the enzymatically produced 2-ketoenduracidine and observed the conversion to L-End, establishing ShMppQ as the final enzyme in L-End biosynthesis. Additionally, we have observed that MppQ has a relatively high affinity for 2-keto-5-guanidinovaleric acid (i.e., 2-ketoarginine), a shunt product of MppP, indicating the potential role of MppQ in increasing the efficiency of L-End biosynthesis by converting 2-ketoarginine back to the starting material, l-arginine. A panel of potential amino-donor substrates was tested for the transamination activity against a saturating concentration of 2-ketoarginine in end-point assays. Most l-Arg was produced with l-ornithine as the donor substrate. Steady-state kinetic analysis of the transamination reaction with l-Orn and 2-ketoarginine shows that the kinetic constants are in line with those for the amino donor substrate of other fold-type I aminotransferases.


Assuntos
Fosfato de Piridoxal , Transaminases , Cinética , Transaminases/metabolismo , Fosfato de Piridoxal/metabolismo , Fosfatos , Especificidade por Substrato , Cristalografia por Raios X
2.
Biochemistry ; 58(40): 4136-4147, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31524380

RESUMO

The acetoacetate decarboxylase-like superfamily (ADCSF) is a little-explored group of enzymes that may contain new biocatalysts. The low level of sequence identity (∼20%) between many ADCSF enzymes and the confirmed acetoacetate decarboxylases led us to investigate the degree of diversity in the reaction and substrate specificity of ADCSF enzymes. We have previously reported on Sbi00515, which belongs to Family V of the ADCSF and functions as an aldolase-dehydratase. Here, we more thoroughly characterize the substrate specificity of Sbi00515 and find that aromatic, unsaturated aldehydes yield lower KM and higher kcat values compared to those of other small electrophilic substrates in the condensation reaction. The roles of several active site residues were explored by site-directed mutagenesis and steady state kinetics. The lysine-glutamate catalytic dyad, conserved throughout the ADCSF, is required for catalysis. Tyrosine 252, which is unique to Sbi00515, is hypothesized to orient the incoming aldehyde in the condensation reaction. Transient state kinetics and an intermediate-bound crystal structure aid in completing a proposed mechanism for Sbi00515.


Assuntos
Aldeído Liases/química , Proteínas de Bactérias/química , Hidroliases/química , Aldeído Liases/genética , Aldeído Liases/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Hidroliases/genética , Hidroliases/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Streptomyces/enzimologia , Especificidade por Substrato
3.
Biochemistry ; 57(23): 3252-3264, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29473729

RESUMO

The PLP-dependent l-arginine hydroxylase/deaminase MppP from Streptomyces wadayamensis (SwMppP) is involved in the biosynthesis of l-enduracididine, a nonproteinogenic amino acid found in several nonribosomally produced peptide antibiotics. SwMppP uses only PLP and molecular oxygen to catalyze a 4-electron oxidation of l-arginine to form a mixture of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid. Steady-state kinetics analysis in the presence and absence of catalase shows that one molecule of peroxide is formed for every molecule of dioxygen consumed in the reaction. Moreover, for each molecule of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid produced, two molecules of dioxygen are consumed, suggesting that both the 4-hydroxy and 2-keto groups are derived from water. This was confirmed by running the reactions using either [18]O2 or H2[18]O and analyzing the products by ESI-MS. Incorporation of [18]O was only observed when the reaction was performed in H2[18]O. Crystal structures of SwMppP with l-arginine, 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid, or 2-oxo-5-guanidinovaleric acid bound were determined at resolutions of 2.2, 1.9. and 1.8 Å, respectively. The structural data show that the N-terminal portion of the protein is disordered unless substrate or product is bound in the active site, in which case it forms a well-ordered helix that covers the catalytic center. This observation suggested that the N-terminal helix may have a role in substrate binding and/or catalysis. Our structural and kinetic characterizations of N-terminal variants show that the N-terminus is critical for catalysis. In light of this new information, we have refined our previously proposed mechanism of the SwMppP-catalyzed oxidation of l-arginine.


Assuntos
Amônia-Liases/química , Proteínas de Bactérias/química , Hidrolases/química , Streptomyces/enzimologia , Arginina/química , Biocatálise , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Domínios Proteicos , Estrutura Secundária de Proteína
4.
Biochemistry ; 54(47): 7029-40, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26551990

RESUMO

L-Enduracididine (L-End) is a nonproteinogenic amino acid found in a number of bioactive peptides, including the antibiotics teixobactin, enduracidin, and mannopeptimycin. The potent activity of these compounds against antibiotic-resistant pathogens like MRSA and their novel mode of action have garnered considerable interest for the development of these peptides into clinically relevant antibiotics. This goal has been hampered, at least in part, by the fact that L-End is difficult to synthesize and not currently commercially available. We have begun to elucidate the biosynthetic pathway of this unusual building block. In mannopeptimycin-producing strains, like Streptomyces wadayamensis, L-End is produced from L-Arg by the action of three enzymes: MppP, MppQ, and MppR. Herein, we report the structural and functional characterization of MppP. This pyridoxal 5'-phosphate (PLP)-dependent enzyme was predicted to be a fold type I aminotransferase on the basis of sequence analysis. We show that MppP is actually the first example of a PLP-dependent hydroxylase that catalyzes a reaction of L-Arg with dioxygen to yield a mixture of 2-oxo-4-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid in a 1.7:1 ratio. The structure of MppP with PLP bound to the catalytic lysine residue (Lys221) shows that, while the tertiary structure is very similar to those of the well-studied aminotransferases, there are differences in the arrangement of active site residues around the cofactor that likely account for the unusual activity of this enzyme. The structure of MppP with the substrate analogue D-Arg bound shows how the enzyme binds its substrate and indicates why D-Arg is not a substrate. On the basis of this work and previous work with MppR, we propose a plausible biosynthetic scheme for L-End.


Assuntos
Arginina/metabolismo , Oxigenases de Função Mista/metabolismo , Fosfato de Piridoxal/metabolismo , Pirrolidinas/metabolismo , Streptomyces/enzimologia , Transaminases/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Vias Biossintéticas , Domínio Catalítico , Cristalografia por Raios X , Guanidinas/metabolismo , Oxigenases de Função Mista/química , Modelos Moleculares , Conformação Proteica , Pirrolidinas/química , Streptomyces/química , Streptomyces/metabolismo , Especificidade por Substrato , Transaminases/química
5.
Biochemistry ; 54(25): 3978-88, 2015 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-26039798

RESUMO

The acetoacetate decarboxylase-like superfamily (ADCSF) is a group of ~4000 enzymes that, until recently, was thought to be homogeneous in terms of the reaction catalyzed. Bioinformatic analysis shows that the ADCSF consists of up to seven families that differ primarily in their active site architectures. The soil-dwelling bacterium Streptomyces bingchenggensis BCW-1 produces an ADCSF enzyme of unknown function that shares a low level of sequence identity (~20%) with known acetoacetate decarboxylases (ADCs). This enzyme, Sbi00515, belongs to the MppR-like family of the ADCSF because of its similarity to the mannopeptimycin biosynthetic protein MppR from Streptomyces hygroscopicus. Herein, we present steady state kinetic data that show Sbi00515 does not catalyze the decarboxylation of any α- or ß-keto acid tested. Rather, we show that Sbi00515 catalyzes the condensation of pyruvate with a number of aldehydes, followed by dehydration of the presumed aldol intermediate. Thus, Sbi00515 is a pyruvate aldolase-dehydratase and not an acetoacetate decarboxylase. We have also determined the X-ray crystal structures of Sbi00515 in complexes with formate and pyruvate. The structures show that the overall fold of Sbi00515 is nearly identical to those of both ADC and MppR. The pyruvate complex is trapped as the Schiff base, providing evidence that the Schiff base chemistry that drives the acetoacetate decarboxylases has been co-opted to perform a new function, and that this core chemistry may be conserved across the superfamily. The structures also suggest possible catalytic roles for several active site residues.


Assuntos
Proteínas de Bactérias/metabolismo , Carboxiliases/química , Frutose-Bifosfato Aldolase/metabolismo , Hidroliases/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Carboxiliases/genética , Carboxiliases/metabolismo , Cristalografia por Raios X , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/genética , Hidroliases/química , Hidroliases/genética , Cetoácidos/metabolismo , Cinética , Ácido Pirúvico/metabolismo , Streptomyces/química , Streptomyces/genética
8.
Biochemistry ; 52(26): 4492-506, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23758195

RESUMO

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus. Enduracididine is produced in Streptomyces hygroscopicus by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5'-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of "4-hydroxy-2-ketoarginine" shows the correct 4R enantiomer of "2-ketoenduracididine" bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function.


Assuntos
Proteínas de Bactérias/química , Carboxiliases/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Streptomyces/enzimologia , Sequência de Aminoácidos , Carboxiliases/classificação , Catálise , Domínio Catalítico , Biologia Computacional/métodos , Cristalografia por Raios X , Peptídeos Cíclicos/biossíntese , Conformação Proteica , Relação Estrutura-Atividade
9.
J Org Chem ; 77(1): 300-10, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22073965

RESUMO

The stereospecific synthesis of aryloxy and amino substituted E- and Z-ethyl-3-acrylates is of interest because of their potential in the polymer industry and in medicinal chemistry. During work on a copper-catalyzed cross-coupling reaction of ethyl (E)- and (Z)-3-iodoacrylates with phenols and N-heterocycles, we discovered a very simple (nonmetallic) method for the stereospecific synthesis of aryloxy and amino substituted acrylates. To study this long-standing problem on the stereoselectivity of aryloxy and amino substituted acrylates, a series of O- and N-substituted nucleophiles was allowed to react with ethyl (E)- and (Z)-3-iodoacrylates. Screening of different bases indicated that DABCO (1,4-diazabicyclo[2.2.2]octane) afforded successful conversion of ethyl (E)- and (Z)-3-iodoacrylates into aryloxy and amino substituted ethyl acrylates in a stereospecific manner. Herein are the details of this DABCO-mediated stereospecific synthesis of aryloxy and amino substituted E- or Z-acrylates.


Assuntos
Acrilatos/síntese química , Cobre/química , Reagentes de Ligações Cruzadas/química , Éteres Fenílicos/síntese química , Polímeros/síntese química , Acrilatos/química , Aminação , Catálise , Estrutura Molecular , Éteres Fenílicos/química , Polímeros/química , Estereoisomerismo
10.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 12): 672-681, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29199988

RESUMO

The Gram-negative bacterium Sphingomonas wittichii RW1 is notable for its ability to metabolize a variety of aromatic hydrocarbons. Not surprisingly, the S. wittichii genome contains a number of putative aromatic hydrocarbon-degrading gene clusters. One of these includes an enzyme of unknown function, Swit_4259, which belongs to the acetoacetate decarboxylase-like superfamily (ADCSF). Here, it is reported that Swit_4259 is a small (28.8 kDa) tetrameric ADCSF enzyme that, unlike the prototypical members of the superfamily, does not have acetoacetate decarboxylase activity. Structural characterization shows that the tertiary structure of Swit_4259 is nearly identical to that of the true decarboxylases, but there are important differences in the fine structure of the Swit_4259 active site that lead to a divergence in function. In addition, it is shown that while it is a poor substrate, Swit_4259 can catalyze the hydration of 2-oxo-hex-3-enedioate to yield 2-oxo-4-hydroxyhexanedioate. It is also demonstrated that Swit_4259 has pyruvate aldolase-dehydratase activity, a feature that is common to all of the family V ADCSF enzymes studied to date. The enzymatic activity, together with the genomic context, suggests that Swit_4259 may be a hydratase with a role in the metabolism of an as-yet-unknown hydrocarbon. These data have implications for engineering bioremediation pathways to degrade specific pollutants, as well as structure-function relationships within the ADCSF in general.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carboxiliases/química , Sphingomonas/enzimologia , Acetoacetatos/química , Acetoacetatos/metabolismo , Proteínas de Bactérias/genética , Carboxiliases/genética , Carboxiliases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ácidos Cetoglutáricos/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato
11.
Org Lett ; 18(17): 4174-7, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27526647

RESUMO

An enolate driven copper-mediated cross-coupling process enabled cheaper and greener access to the key pentacyclic intermediates required for the enantiospecific total synthesis of a number of C-19 methyl substituted sarpagine/macroline indole alkaloids. Replacement of palladium (60-68%) with copper iodide (82-89%) resulted in much higher yields. The formation of an unusual 7-membered cross-coupling product was completely inhibited by using TEMPO as a radical scavenger. Further functionalization led to the first enantiospecific total synthesis of macrocarpines D and E.


Assuntos
Cobre/química , Iodetos/química , Paládio/química , Floroglucinol/síntese química , Catálise , Conformação Molecular , Floroglucinol/análogos & derivados , Floroglucinol/química , Estereoisomerismo
12.
J Org Chem ; 64(24): 8922-8928, 1999 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-11674799

RESUMO

Ligand 1, bearing two ethylenediamine groups, was designed to form a hydrophobic cavity upon binding to metals. The shape of its nonpolar cavity depends on the metal: a reversal in binding preference for naphthalene or biphenyl groups is found when the metal is changed from zinc to copper, with a selectivity change of 260-fold. In the presence of dansylated amino acids, the new ligand constitutes a fluorescent sensor for zinc ion. Variations are seen in affinity for dansylamino acid with minor structural changes, and organic selectivity changes with complexes of variant metals. These findings suggest that sensor tuning of affinity and selectivity for metal is possible by choice of simple organic guest and for organic guest by choice of metal.

13.
J Org Chem ; 64(6): 1784-1788, 1999 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-11674265

RESUMO

Diarylphosphinic acid 2, bearing two ethylenediamine groups, was designed with the aim of assembly to form a hydrophobic cavity upon binding to metals. The new ligand was prepared in optically active form from phenylalanine by an efficient sequence which required chromatography only for the P-C bond forming steps. Versatile intermediates in the synthesis are described. Complexes of this ligand with Zn(2+) were shown to bind closely related aromatic guests bearing anionic tethered groups with 1000-fold discrimination.

15.
J Comb Chem ; 8(2): 221-7, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16529517

RESUMO

Sensor arrays are useful for many purposes. Our interests include quasi-distributed intrinsic fiber optic arrays, those distributed along the length of an optical fiber. We have demonstrated an optical time-of-flight approach to distinguishing the fluorescence output of such arrays, as well as a synthesis of combinatorial libraries that takes advantage of a support of linear morphology to make numerous compounds in a simple manner without information loss in the synthesis. To unite these research areas, we needed an optical fiber cladding material that meets demanding synthetic and optical requirements. We have chosen the Meldal SPOCC polymer support as the best candidate for such a material and report here our initial results with this material.


Assuntos
Técnicas de Química Combinatória/instrumentação , Técnicas de Química Combinatória/métodos , Flúor , Polímeros/síntese química , Tecnologia de Fibra Óptica , Fibras Ópticas , Piperidinas , Polietilenoglicóis
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA