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1.
Elife ; 122023 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-36881526

RESUMO

Malonyl-CoA-acyl carrier protein transacylase (MCAT) is an enzyme involved in mitochondrial fatty acid synthesis (mtFAS) and catalyzes the transfer of the malonyl moiety of malonyl-CoA to the mitochondrial acyl carrier protein (ACP). Previously, we showed that loss-of-function of mtFAS genes, including Mcat, is associated with severe loss of electron transport chain (ETC) complexes in mouse immortalized skeletal myoblasts (Nowinski et al., 2020). Here, we report a proband presenting with hypotonia, failure to thrive, nystagmus, and abnormal brain MRI findings. Using whole exome sequencing, we identified biallelic variants in MCAT. Protein levels for NDUFB8 and COXII, subunits of complex I and IV respectively, were markedly reduced in lymphoblasts and fibroblasts, as well as SDHB for complex II in fibroblasts. ETC enzyme activities were decreased in parallel. Re-expression of wild-type MCAT rescued the phenotype in patient fibroblasts. This is the first report of a patient with MCAT pathogenic variants and combined oxidative phosphorylation deficiency.


Assuntos
Proteína de Transporte de Acila S-Maloniltransferase , Doenças Mitocondriais , Animais , Camundongos , Adipogenia , Encéfalo , Mitocôndrias , Doenças Mitocondriais/genética , Proteína de Transporte de Acila S-Maloniltransferase/genética
2.
J Agric Food Chem ; 70(43): 13922-13934, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36264009

RESUMO

The malonyl-CoA:ACP transacylase (MAT) domain is responsible for the selection and incorporation of malonyl building blocks in the biosynthesis of polyunsaturated fatty acids (PUFAs) in eukaryotic microalgae (Schizochytrium) and marine bacteria (Moritella marina, Photobacterium profundum, and Shewanella). Elucidation of the structural basis underlying the substrate specificity and catalytic mechanism of the MAT will help to improve the yield and quality of PUFAs. Here, a methodology guided by molecular dynamics simulations was carried out to identify and mutate specificity-conferring residues within the MAT domain of Schizochytrium. Combining mutagenesis, cell-free protein synthesis, and in vitro biochemical assay, we dissected nearby interactions and molecular mechanisms relevant for binding and catalysis and found that the reorientation of the Ser154 Cß-Oγ bond establishes distinctive proton-transfer chains (His153-Ser154 and Asn235-His153-Ser154) for catalysis. Gln66 can be replaced by tyrosine to shorten the distance between His153 (Nε2) and Ser154 (Oγ), which facilitates a faster proton-transfer rate, allowing better use of acyl substrates than the wild type. Furthermore, we screened a mutant that displayed an 18.4% increase in PUFA accumulation. These findings provide important insights into the study of MAT through protein engineering and will benefit dissecting the molecular mechanisms of other PUFA-related catalytic domains.


Assuntos
Malonil Coenzima A , Estramenópilas , Malonil Coenzima A/metabolismo , Prótons , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Aciltransferases/metabolismo , Estramenópilas/metabolismo , Ácidos Graxos Insaturados/metabolismo
3.
Genes (Basel) ; 12(4)2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33918393

RESUMO

Pathological variants in the nuclear malonyl-CoA-acyl carrier protein transacylase (MCAT) gene, which encodes a mitochondrial protein involved in fatty-acid biogenesis, have been reported in two siblings from China affected by insidious optic nerve degeneration in childhood, leading to blindness in the first decade of life. After analysing 51 families with negative molecular diagnostic tests, from a cohort of 200 families with hereditary optic neuropathy (HON), we identified two novel MCAT mutations in a female patient who presented with acute, sudden, bilateral, yet asymmetric, central visual loss at the age of 20. This presentation is consistent with a Leber hereditary optic neuropathy (LHON)-like phenotype, whose existence and association with NDUFS2 and DNAJC30 has only recently been described. Our findings reveal a wider phenotypic presentation of MCAT mutations, and a greater genetic heterogeneity of nuclear LHON-like phenotypes. Although MCAT pathological variants are very uncommon, this gene should be investigated in HON patients, irrespective of disease presentation.


Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/genética , Mutação , Atrofia Óptica Hereditária de Leber/genética , Análise de Sequência de DNA/métodos , Proteína de Transporte de Acila S-Maloniltransferase/química , Feminino , França , Humanos , Modelos Moleculares , Linhagem , Conformação Proteica , Adulto Jovem
4.
Proc Natl Acad Sci U S A ; 117(39): 24224-24233, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32929027

RESUMO

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.


Assuntos
Proteína de Transporte de Acila/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Domínio Catalítico , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Cristalografia por Raios X
5.
J Biol Chem ; 295(22): 7743-7752, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32341123

RESUMO

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9-assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.


Assuntos
Apicoplastos/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Toxoplasma/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/genética , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Apicoplastos/genética , Ácidos Graxos/genética , Deleção de Genes , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética
6.
Hum Mol Genet ; 29(3): 444-458, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31915829

RESUMO

Inherited optic neuropathies are rare eye diseases of optic nerve dysfunction that present in various genetic forms. Previously, mutation in three genes encoding mitochondrial proteins has been implicated in autosomal recessive forms of optic atrophy that involve progressive degeneration of optic nerve and retinal ganglion cells (RGC). Using whole exome analysis, a novel double homozygous mutation p.L81R and pR212W in malonyl CoA-acyl carrier protein transacylase (MCAT), a mitochondrial protein involved in fatty acid biosynthesis, has now been identified as responsible for an autosomal recessive optic neuropathy from a Chinese consanguineous family. MCAT is expressed in RGC that are rich in mitochondria. The disease variants lead to structurally unstable MCAT protein with significantly reduced intracellular expression. RGC-specific knockdown of Mcat in mice, lead to an attenuated retinal neurofiber layer, that resembles the phenotype of optic neuropathy. These results indicated that MCAT plays an essential role in mitochondrial function and maintenance of RGC axons, while novel MCAT p.L81R and p.R212W mutations can lead to optic neuropathy.


Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/genética , Genes Recessivos , Mitocôndrias/patologia , Doenças do Nervo Óptico/patologia , Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Proteína de Transporte de Acila S-Maloniltransferase/química , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Sequência de Aminoácidos , Animais , Criança , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mutação , Nervo Óptico/metabolismo , Doenças do Nervo Óptico/etiologia , Doenças do Nervo Óptico/metabolismo , Linhagem , Conformação Proteica , Células Ganglionares da Retina/metabolismo , Homologia de Sequência , Sequenciamento do Exoma
7.
Methods Mol Biol ; 1927: 23-35, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788783

RESUMO

Antisense RNA (asRNA) technology is an important tool for downregulating gene expression. When applying this strategy, the asRNA interference efficiency is determined by several elements including scaffold design, loop size, and relative abundance. Here, we take the Escherichia coli gene fabD encoding malonyl-CoA-[acyl-carrier-protein] transacylase as an example to describe the asRNA design with reliable and controllable interference efficiency. Real-time PCR and fluorescence assay methods are introduced to detect the interference efficiency at RNA level and protein level, respectively.


Assuntos
Regulação da Expressão Gênica , RNA Antissenso/genética , Proteína de Transporte de Acila S-Maloniltransferase/genética , Regulação para Baixo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ácido Graxo Sintase Tipo II/genética , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Plasmídeos/genética , Interferência de RNA , RNA Antissenso/química , Reação em Cadeia da Polimerase em Tempo Real/métodos
8.
Plant Cell Physiol ; 60(6): 1239-1249, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30796840

RESUMO

Malonyl-acyl carrier protein (ACP) is a key building block for the synthesis of fatty acids, which are important components of cell membranes, storage oils and lipid-signaling molecules. Malonyl CoA-ACP malonyltransferase (MCAMT) catalyzes the production of malonyl-ACP and CoA from malonyl-CoA and ACP. Here, we report that MCAMT plays a critical role in cell division and has the potential to increase the storage oil content in Arabidopsis. The quantitative real-time PCR and MCAMT promoter:GUS analyses showed that MCAMT is predominantly expressed in shoot and root apical meristems, leaf hydathodes and developing embryos. The fluorescent signals of MCAMT:eYFP were observed in both chloroplasts and mitochondria of tobacco leaf protoplasts. In particular, the N-terminal region (amino acid residues 1-30) of MCAMT was required for mitochondrial targeting. The Arabidopsis mcamt-1 and -2 mutants exhibited an embryo-lethal phenotype because of the arrest of embryo development at the globular stage. The transgenic Arabidopsis expressing antisense MCAMT RNA showed growth retardation caused by the defects in cell division. The overexpression of MCAMT driven by the promoter of the senescence-associated 1 (SEN1) gene, which is predominantly expressed in developing seeds, increased the seed yield and storage oil content of Arabidopsis. Taken together, the plastidial and mitochondrial MCAMT is essential for Arabidopsis cell division and is a novel genetic resource useful for enhancing storage oil content in oilseed crops.


Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Proteínas de Arabidopsis/metabolismo , Divisão Celular , Mitocôndrias/enzimologia , Óleos de Plantas/metabolismo , Plastídeos/enzimologia , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/metabolismo
9.
J Agric Food Chem ; 66(51): 13444-13453, 2018 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-30488696

RESUMO

Resveratrol, a plant-derived polyphenolic compound with various health activities, is widely used in nutraceutical and food additives. Herein, combinatorial optimization of resveratrol biosynthetic pathway and intracellular environment of E. coli was carried out. By screening pathway genes from various species and exploring their expression pattern, we initially constructed resveratrol-producing strains. Further targeting at availability of malonyl-CoA through expressing ACC of Corynebacterium glutamicum and antisense inhibiting native fabD significantly increased resveratrol biosynthesis. Transport engineering for resveratrol secretion and molecular chaperones helping for folding heterologous enzymes were employed to improve the intracellular environments in remarkable degrees. By introducing PcTAL of Phanerochaete chrysosporium and tuning expression model of PcTAL, At4CL, and VvSTS, an engineered E. coli produced 57.77 mg/L of resveratrol from l-tyrosine. After integrating the above strategies, resveratrol titer reached to 238.71 mg/L from l-tyrosine. The combinatorial optimization in this study provides a promising strategy to produce valuable natural products in heterologous expression systems.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Resveratrol/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/genética , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Vias Biossintéticas , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Malonil Coenzima A/genética , Malonil Coenzima A/metabolismo , Phanerochaete/enzimologia , Phanerochaete/genética
10.
Biochim Biophys Acta Proteins Proteom ; 1866(11): 1131-1142, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30282611

RESUMO

The fatty acid biosynthesis (FAS II) is a vital process in bacteria and regarded as an attractive pathway for the development of potential antimicrobial agents. In this study, we report 1,4-naphthoquinone (NPQ) as a dual inhibitor of two key enzymes of FAS II pathway, namely FabD (Malonyl-CoA:ACP transacylase) and FabZ (ß-hydroxyacyl-ACP dehydratase). Mode of inhibition of NPQ was found to be non-competitive for both enzymes with IC50 of 26.67 µΜ and 23.18 µΜ against McFabZ and McFabD respectively. Conformational changes in secondary and tertiary structures marked by the loss of helical contents were observed in both enzymes upon NPQ binding. The fluorescence quenching was found to be static with a stable ground state complex formation. ITC based studies have shown that NPQ is binding to McFabZ with a stronger affinity (~1.5×) as compared to McFabD. Molecular docking studies have found that NPQ interacts with key residues of both McFabD (Ser209, Arg126, and Leu102) and McFabZ (His74 and Tyr112) enzymes. Both complexes have shown the structural stability during the 20 ns run of molecular dynamics based simulations. Altogether, the present study suggests that NPQ scaffold can be exploited as a multi-targeted inhibitor of FAS II pathway, and these biochemical and biophysical findings will further help in the development of potent antibacterial agents targeting FAS II pathway.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Moraxella catarrhalis/enzimologia , Naftoquinonas/farmacologia , Proteína de Transporte de Acila S-Maloniltransferase/antagonistas & inibidores , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Dicroísmo Circular , Malonil Coenzima A/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Moraxella catarrhalis/efeitos dos fármacos , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termodinâmica
11.
Biochem Biophys Res Commun ; 505(1): 208-214, 2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30243724

RESUMO

Bacterial fatty acid synthesis (FAS) has been extensively studied as a potential target of antimicrobials. In FAS, FabD mediates transacylation of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP). The mounting threat of nosocomial infection by multidrug-resistant Acinetobacter baumannii warrants a deeper understanding of its essential cellular mechanisms, which could lead to effective control of this highly competent pathogen. The molecular mechanisms involved in A. baumannii FAS are poorly understood, and recent research has suggested that Pseudomonas aeruginosa, a closely related nosocomial pathogen of A. baumannii, utilizes FAS to produce virulence factors. In this study, we solved the crystal structure of A. baumannii FabD (AbFabD) to provide a platform for the development of new antibacterial agents. Analysis of the structure of AbFabD confirmed the presence of highly conserved active site residues among bacterial homologs. Binding constants between AbFabD variants and A. baumannii ACP (AbACP) revealed critical conserved residues Lys195 and Lys200 involved in AbACP binding. Computational docking of a potential inhibitor, trifluoperazine, revealed a unique inhibitor-binding pocket near the substrate-binding site. The structural study presented herein will be useful for the structure-based design of potent AbFabD inhibitors.


Assuntos
Acinetobacter baumannii/genética , Proteína de Transporte de Acila S-Maloniltransferase/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Ácido Graxo Sintase Tipo II/genética , Acinetobacter baumannii/enzimologia , Proteína de Transporte de Acila S-Maloniltransferase/química , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/metabolismo , Modelos Moleculares , Mutação , Domínios Proteicos , Homologia de Sequência de Aminoácidos
12.
Appl Microbiol Biotechnol ; 102(15): 6333-6341, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29858956

RESUMO

This review will cover the structure, enzymology, and related aspects that are important for structure-based engineering of the transacylase enzymes from fatty acid biosynthesis and polyketide synthesis. Furthermore, this review will focus on in vitro characteristics and not cover engineering of the upstream or downstream reactions or strategies to manipulate metabolic flux in vivo. The malonyl-coenzyme A(CoA)-holo-acyl-carrier protein (holo-ACP) transacylase (FabD) from Escherichia coli serves as a model for this enzyme with thorough descriptions of structure, enzyme mechanism, and effects of mutation on substrate binding presented in the literature. Here, we discuss multiple practical and theoretical considerations regarding engineering transacylase enzymes to accept non-cognate substrates and form novel acyl-ACPs for downstream reactions.


Assuntos
Acil Coenzima A/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/genética , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Bioengenharia/tendências , Microbiologia Industrial/métodos , Acil Coenzima A/genética , Proteína de Transporte de Acila S-Maloniltransferase/química , Aciltransferases/química , Aciltransferases/genética , Aciltransferases/metabolismo , Microbiologia Industrial/tendências
13.
J Agric Food Chem ; 66(21): 5382-5391, 2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-29722541

RESUMO

Polyunsaturated fatty acids (PUFAs) have been widely applied in the food and medical industry. In this study, malonyl-CoA: ACP transacylase (MAT) was overexpressed through homologous recombination to improve PUFA production in Schizochytrium. The results showed that the lipid and PUFA concentration were increased by 10.1 and 24.5% with MAT overexpression, respectively. Metabolomics analysis revealed that the intracellular tricarboxylic acid cycle was weakened and glucose absorption was accelerated in the engineered strain. In the mevalonate pathway, intracellular carotene content was decreased, and the carbon flux was then redirected toward PUFA synthesis. Furthermore, a glucose fed-batch fermentation was finally performed with the engineered Schizochytrium. The total lipid yield was further increased to 110.5 g/L, 39.6% higher than the wild strain. Docosahexaenoic acid and eicosapentaenoic acid yield were enhanced to 47.39 g/L and 1.65 g/L with an increase of 81.5 and 172.5%, respectively. This study provided an effective metabolic engineering strategy for industrial PUFA production.


Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/genética , Ácidos Graxos Insaturados/biossíntese , Expressão Gênica , Estramenópilas/metabolismo , Ciclo do Ácido Cítrico , Ácido Eicosapentaenoico/biossíntese , Fermentação , Glucose/metabolismo , Recombinação Homóloga/genética , Metabolômica , Estramenópilas/genética
14.
Biochimie ; 149: 18-33, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29604333

RESUMO

Malonyl-CoA:acyl carrier protein transacylase (FabD), being an essential enzyme of the FAS II pathway, is an attractive target for developing broad-spectrum antibiotics. It performs initiation reaction to form malonyl-ACP, which is a key building block in fatty acid biosynthesis. In this study, we have characterized the FabD from drug-resistant pathogen Moraxella catarrhalis (McFabD). More importantly, we have shown the binding of McFabD with three new compounds from the class of aporphine alkaloids. ITC based binding studies have shown that apomorphine is binding to McFabD with a stronger affinity (KD = 4.87 µM) as compared to boldine (KD = 7.19 µM) and magnoflorine (KD = 11.7 µM). The possible mechanism of fluorescence quenching is found to be static with Kq values higher than 1010, which was associated with the ground state complex formation of aporphine alkaloids with McFabD. Conformational changes observed in the secondary and tertiary structure marked by the loss of helical content during the course of interactions. Molecular docking based studies have predicted the binding mode of aporphine alkaloids and it is found that these compounds are interacting in a similar fashion as known inhibitor corytuberine is interacting with McFabD. The analysis of docking poses have revealed that His 210, Leu102, Gln19, Ser101 and Arg 126 are critical residues, which may play important role in binding. The growth inhibition assay has shown that apomorphine has better MIC value (4-8 µg/ml) against Moraxella catarrhalis as compared to boldine and magnoflorine. Therefore, the current study suggests that aporphine alkaloids can act as antibacterial agents and possible target of these compounds could be FabD enzyme from the FAS II pathway, and apomorphine scaffold will be more suitable among these compounds for potential development of antibacterial agents.


Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/química , Alcaloides/química , Aporfinas/química , Moraxella catarrhalis/química , Alcaloides/farmacologia , Aporfinas/farmacologia , Fenômenos Biofísicos , Simulação por Computador , Resistência Microbiana a Medicamentos/genética , Humanos , Simulação de Acoplamento Molecular , Moraxella catarrhalis/efeitos dos fármacos , Moraxella catarrhalis/crescimento & desenvolvimento , Moraxella catarrhalis/patogenicidade , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos
15.
Appl Microbiol Biotechnol ; 101(23-24): 8431-8441, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29075826

RESUMO

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and ß-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min-1 mg-1. FabD R117A maintained K M values for holo-ACP (~ 40 µM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 µM) and acetyl-CoA (200 ± 70 µM) when compared to malonyl-CoA (80 ± 30 µM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.


Assuntos
Proteína de Transporte de Acila/biossíntese , Proteína de Transporte de Acila S-Maloniltransferase/genética , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/biossíntese , Cinética
16.
BMC Struct Biol ; 17(1): 1, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28143508

RESUMO

BACKGROUND: The post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa. While the disordered behavior of free Pup has been well characterized, it remained unknown whether Pup adopts a distinct structure when attached to a substrate. RESULTS: Using a combination of NMR experiments and biochemical analysis we demonstrate that Pup remains unstructured when ligated to two well-established pupylation substrates targeted for proteasomal degradation in Mycobacterium tuberculosis, malonyl transacylase (FabD) and ketopantoyl hydroxylmethyltransferase (PanB). Isotopically labeled Pup was linked to FabD and PanB by in vitro pupylation to generate homogeneously pupylated substrates suitable for NMR analysis. The single target lysine of PanB was identified by a combination of mass spectroscopy and mutational analysis. Chemical shift comparison between Pup in its free form and ligated to substrate reveals intrinsic disorder of Pup in the conjugate. CONCLUSION: When linked to the proteasomal substrates FabD and PanB, Pup is unstructured and retains the ability to interact with its different binding partners. This suggests that it is not the conformation of Pup attached to these two substrates which determines their delivery to the proteasome, but the availability of the degradation complex and the depupylase.


Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/química , Proteínas de Bactérias/química , Hidroximetil e Formil Transferases/química , Mycobacterium tuberculosis/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/química , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Hidroximetil e Formil Transferases/metabolismo , Modelos Moleculares , Conformação Proteica , Proteólise , Especificidade por Substrato , Ubiquitinação , Ubiquitinas/metabolismo
17.
Biotechnol Appl Biochem ; 64(5): 620-626, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27572053

RESUMO

Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to the lack of suitable industrial strains with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT overexpressing N. oceanica showed a higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. Gas chromatography-mass spectrometry analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of eicosapentaenoic acid (C20:5), which is a polyunsaturated fatty acid necessary for animal nutrition, increased by 8%. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential.


Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/genética , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Ácidos Graxos/metabolismo , Microalgas/enzimologia , Estramenópilas/enzimologia , Proteína de Transporte de Acila S-Maloniltransferase/classificação , Biocombustíveis , Ácidos Graxos/análise , Microalgas/genética , Microalgas/metabolismo , Estramenópilas/genética , Estramenópilas/metabolismo
18.
Nat Commun ; 7: 12944, 2016 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-27703138

RESUMO

The bacterial pathway for fatty acid biosynthesis, FASII, is a target for development of new anti-staphylococcal drugs. This strategy is based on previous reports indicating that self-synthesized fatty acids appear to be indispensable for Staphylococcus aureus growth and virulence, although other bacteria can use exogenous fatty acids to compensate FASII inhibition. Here we report that staphylococci can become resistant to the FASII-targeted inhibitor triclosan via high frequency mutations in fabD, one of the FASII genes. The fabD mutants can be conditional for FASII and not require exogenous fatty acids for normal growth, and can use diverse fatty acid combinations (including host fatty acids) when FASII is blocked. These mutants show cross-resistance to inhibitors of other FASII enzymes and are infectious in mice. Clinical isolates bearing fabD polymorphisms also bypass FASII inhibition. We propose that fatty acid-rich environments within the host, in the presence of FASII inhibitors, might favour the emergence of staphylococcal strains displaying resistance to multiple FASII inhibitors.


Assuntos
Farmacorresistência Bacteriana , Ácidos Graxos/metabolismo , Mutação , Staphylococcus aureus/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Alelos , Animais , Antibacterianos/farmacologia , Clonagem Molecular , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Feminino , Teste de Complementação Genética , Lipogênese , Camundongos , Camundongos Endogâmicos BALB C , Polimorfismo Genético , Análise de Sequência de DNA , Triclosan/farmacologia , Virulência/efeitos dos fármacos
19.
Chembiochem ; 17(22): 2137-2142, 2016 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-27598417

RESUMO

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl-CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl-CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self-malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self-acylation. This lack of self-malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein-protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Ácido Graxo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/genética , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Antibacterianos/análise , Antibacterianos/química , Proteínas de Bactérias/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Ácido Graxo Sintases/genética , Família Multigênica , Pantoea/enzimologia , Pantoea/genética , Polienos/análise , Polienos/química , Polienos/metabolismo , Policetídeo Sintases/genética , Domínios e Motivos de Interação entre Proteínas , Pirróis/análise , Pirróis/química , Pirróis/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
World J Microbiol Biotechnol ; 32(6): 102, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27116968

RESUMO

Pinosylvin as a bioactive stilbene is of great interest for food supplements and pharmaceuticals development. In comparison to conventional extraction of pinosylvin from plant sources, biosynthesis engineering of microbial cell factories is a sustainable and flexible alternative method. Current synthetic strategies often require expensive phenylpropanoic precursor and inducer, which are not available for large-scale fermentation process. In this study, three bioengineering strategies were described to the development of a simple and economical process for pinosylvin biosynthesis in Escherichia coli. Firstly, we evaluated different construct environments to give a highly efficient constitutive system for enzymes of pinosylvin pathway expression: 4-coumarate: coenzyme A ligase (4CL) and stilbene synthase (STS). Secondly, malonyl coenzyme A (malonyl-CoA) is a key precursor of pinosylvin bioproduction and at low level in E. coli cell. Thus clustered regularly interspaced short palindromic repeats interference (CRISPRi) was explored to inactivate malonyl-CoA consumption pathway to increase its availability. The resulting pinosylvin content in engineered E. coli was obtained a 1.9-fold increase depending on the repression of fabD (encoding malonyl-CoA-ACP transacylase) gene. Eventually, a phenylalanine over-producing E. coli consisting phenylalanine ammonia lyase was introduced to produce the precursor of pinosylvin, trans-cinnamic acid, the crude extraction of cultural medium was used as supplementation for pinosylvin bioproduction. Using these combinatorial processes, 47.49 mg/L pinosylvin was produced from glycerol.


Assuntos
Bioengenharia/métodos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Estilbenos/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/biossíntese , Proteína de Transporte de Acila S-Maloniltransferase/genética , Aciltransferases/metabolismo , Cinamatos/química , Coenzima A Ligases/metabolismo , Ácidos Cumáricos/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Ácido Graxo Sintase Tipo II/biossíntese , Ácido Graxo Sintase Tipo II/genética , Ácidos Graxos/biossíntese , Glicerol/metabolismo , Malonil Coenzima A/metabolismo , Fenilalanina/metabolismo , Estilbenos/química , Estilbenos/economia
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