RESUMEN
Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.
Asunto(s)
Proteínas Fúngicas/metabolismo , Lipasa/metabolismo , Lipólisis , Yarrowia/enzimología , Secuencia de Aminoácidos , Ácidos y Sales Biliares/metabolismo , Cristalografía por Rayos X , Estabilidad de Enzimas , Ácidos Grasos no Esterificados/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glicerol/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Lipasa/química , Lipasa/genética , Modelos Moleculares , Datos de Secuencia Molecular , Aceite de Oliva , Pichia/enzimología , Pichia/genética , Aceites de Plantas/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato , Triglicéridos/metabolismo , Yarrowia/genéticaRESUMEN
BACKGROUND & AIMS: Pancreatic exocrine insufficiency (PEI) reduces pancreatic secretion of digestive enzymes, including lipases. Oral pancreatic enzyme replacement therapy (PERT) with pancreatin produces unsatisfactory results. The lipase 2 produced by the yeast Yarrowia lipolytica (YLLIP2; GenBank: AJ012632) might be used in PERT. We investigated its ability to digest triglycerides in a test meal and its efficacy in reducing fecal fat in an animal model of PEI. METHODS: YLLIP2 was produced by genetically engineered Y lipolytica and purified from culture media. YLLIP2 or other gastric (LIPF) and pancreatic (PNLIPD) lipases were added to a meal paste containing dietary triglycerides, at a range of pH values (pH 2-7), with and without pepsin or human bile and incubated at 37°C. We collected samples at various time points and measured lipase activities and stabilities. To create an animal model of PEI, steatorrhea was induced by embolization of the exocrine pancreas gland and pancreatic duct ligation in minipigs. The animals were given YLLIP2 (1, 4, 8, 40, or 80 mg/d) or pancreatin (100,000 US Pharmacopeia lipase units/d, controls) for 9 days. We then collected stool samples, measured fat levels, and calculated coefficient of fat absorption (CFA) values. RESULTS: YLLIP2 was highly stable and poorly degraded by pepsin, and had the highest activity of all lipases tested on meal triglyceride at pH 4-7 (pH 6 with bile: 94 ± 34 U/mg; pH 4 without bile: 43 ± 13 U/mg). Only gastric lipase was active and stable at pH 3, whereas YLLIP2 was sensitive to pepsin hydrolysis after pH inactivation. From in vitro test meal experiments, the lipase activity of YLLIP2 (10 mg) was estimated to be equivalent to that of pancreatin (1200 mg; 100,000 US Pharmacopeia units) at pH 6. In PEI minipigs, CFA values increased from 60.1% ± 9.3% before surgery to 90.5% ± 3.2% after administration of 1200 mg pancreatin (P < .05); CFA values increased to a range of 84.6% ± 3.0% to 90.0% ± 3.8% after administration of 4-80 mg YLLIP2 (P < .05). CONCLUSIONS: The yeast lipase YLLIP2 is stable and has high levels of activity against test meal triglycerides in a large pH range, with and without bile. Oral administration of milligram amounts of YLLIP2 significantly increased CFA values, similar to that of 1.2 g pancreatin, in a minipig model of PEI.
Asunto(s)
Hidrolasas de Éster Carboxílico/farmacología , Terapia de Reemplazo Enzimático , Insuficiencia Pancreática Exocrina/tratamiento farmacológico , Proteínas Fúngicas/farmacología , Absorción Intestinal/efectos de los fármacos , Lipasa/farmacología , Lipólisis/efectos de los fármacos , Triglicéridos/metabolismo , Yarrowia/enzimología , Administración Oral , Animales , Hidrolasas de Éster Carboxílico/biosíntesis , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Modelos Animales de Enfermedad , Perros , Estabilidad de Enzimas , Insuficiencia Pancreática Exocrina/enzimología , Heces/química , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Lipasa/biosíntesis , Lipasa/genética , Lipasa/aislamiento & purificación , Pancreatina/farmacología , Pepsina A/metabolismo , Proteínas Recombinantes/farmacología , Porcinos , Porcinos Enanos , Factores de Tiempo , Triglicéridos/administración & dosificación , Yarrowia/genéticaRESUMEN
Cutinases are extracellular enzymes that are able to degrade cutin, a polyester protecting plant leaves and many kinds of lipids. Although cutinases are mainly found in phytopathogenic fungi or bacteria, 7 genes related to the cutinase family have been predicted in the genome of Mycobacterium tuberculosis. These genes may encode proteins that are involved in the complex lipid metabolism of the bacterium. Here, we report on the biochemical characterization of two secreted proteins of M. tuberculosis, Rv1984c and Rv3452, belonging to the cutinase family. Although their amino acid sequence shows 50% identity with that of the well-characterized cutinase from Fusarium solani pisi, and a high level of homology has been found to exist between these two enzymes, they show distinct substrate specificities. Rv1984c preferentially hydrolyzes medium-chain carboxylic esters and monoacylglycerols, whereas Rv3452 behaves like a phospholipase A(2), and it is able to induce macrophage lysis. The tetrahydrolipstatin inhibitor, a specific lipase inhibitor, abolishes the activity of both enzymes. Site-directed mutagenesis was performed to identify the catalytic triad of Rv1984c. Structural models for Rv1984c and Rv3452 were built, based on the crystal structure of F. solani cutinase, with a view to investigating the contribution of specific residues to the substrate specificity. Our findings open new prospects for investigating the physiological roles of cutinase-like proteins in the lipid metabolism and virulence of M. tuberculosis.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Ésteres/metabolismo , Lipólisis/fisiología , Mycobacterium tuberculosis/enzimología , Fosfolipasas A2/metabolismo , Secuencia de Aminoácidos , Animales , Hidrolasas de Éster Carboxílico/genética , Catálisis , Inhibidores Enzimáticos/farmacología , Lactonas/farmacología , Lipasa/antagonistas & inhibidores , Lipólisis/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Orlistat , Fosfolipasas A2/genética , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
In this study, we report the DNA sequence and biological analysis of a mycobacterial mercury resistance operon encoding a novel Hg(2+) transporter. MerH was found to transport mercuric ions in Escherichia coli via a pair of essential cysteine residues but only when coexpressed with the mercuric reductase.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Farmacorresistencia Microbiana/genética , Compuestos de Mercurio/metabolismo , Compuestos de Mercurio/farmacología , Mycobacterium marinum/efectos de los fármacos , Mycobacterium marinum/genética , Operón/genética , Secuencia de Aminoácidos , Secuencia Conservada , Cartilla de ADN , Escherichia coli/genética , Datos de Secuencia Molecular , Plásmidos , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Microbial exopolysaccharides (EPSs) play key roles in plant-microbe interactions, such as biofilm formation on plant roots and legume nodulation by rhizobia. Here, we focused on the function of an EPS produced by Rhizobium sp. YAS34 in the colonization and biofilm formation on non-legume plant roots (Arabidopsis thaliana and Brassica napus). Using random transposon mutagenesis, we isolated an EPS-deficient mutant of strain YAS34 impaired in a glycosyltransferase gene (gta). Wild type and mutant strains were tagged with a plasmid-born GFP and, for the first time, the EPS produced by the wild-type strain was seen in the rhizosphere using selective carbohydrate probing with a fluorescent lectin and confocal laser-scanning microscopy. We show for the fist time that Rhizobium forms biofilms on roots of non-legumes, independently of the EPS synthesis. When produced by strain YAS34 wild type, EPS is targeted at specific parts of the plant root system. Nutrient fluctuations, root exudates and bacterial growth phase can account for such a production pattern. The EPS synthesis in Rhizobium sp. YAS34 is not essential for biofilm formation on roots, but is critical to colonization of the basal part of the root system and increasing the stability of root-adhering soil. Thus, in Rhizobium sp. YAS34 and non-legume interactions, microbial EPS is implicated in root-soil interface, root colonization, but not in biofilm formation.
Asunto(s)
Arabidopsis/microbiología , Biopelículas , Brassica napus/microbiología , Polisacáridos Bacterianos/fisiología , Rhizobium/metabolismo , Raíces de Plantas/microbiologíaRESUMEN
Heavy metals such as cadmium (Cd(2+)) affect microbial metabolic processes. Consequently, bacteria adapt by adjusting their cellular machinery. We have investigated the dose-dependent growth effects of Cd(2+) on Rhizobium alamii, an exopolysaccharide (EPS)-producing bacterium that forms a biofilm on plant roots. Adsorption isotherms show that the EPS of R. alamii binds cadmium in competition with calcium. A metabonomics approach based on ion cyclotron resonance Fourier transform mass spectrometry has showed that cadmium alters mainly the bacterial metabolism in pathways implying sugars, purine, phosphate, calcium signalling and cell respiration. We determined the influence of EPS on the bacterium response to cadmium, using a mutant of R. alamii impaired in EPS production (MSΔGT). Cadmium dose-dependent effects on the bacterial growth were not significantly different between the R. alamii wild type (wt) and MSΔGT strains. Although cadmium did not modify the quantity of EPS isolated from R. alamii, it triggered the formation of biofilm vs planktonic cells, both by R. alamii wt and by MSΔGT. Thus, it appears that cadmium toxicity could be managed by switching to a biofilm way of life, rather than producing EPS. We conclude that modulations of the bacterial metabolism and switching to biofilms prevails in the adaptation of R. alamii to cadmium. These results are original with regard to the conventional role attributed to EPS in a biofilm matrix, and the bacterial response to cadmium.
Asunto(s)
Biopelículas/efectos de los fármacos , Cadmio/toxicidad , Polisacáridos Bacterianos/biosíntesis , Rhizobium/efectos de los fármacos , Rhizobium/metabolismo , Rhizobium/genética , Rhizobium/crecimiento & desarrolloRESUMEN
Mercuric ion resistance in bacteria requires transport of mercuric ions (Hg(2+)) into the cytoplasmic compartment where they are reduced to the less toxic metallic mercury (Hg(0)) by mercuric reductase (MR). The long-established model for the resistance mechanism predicts interactions between the inner membrane mercuric ion transporter, MerT, and the N-terminal domain of cytoplasmic MR, but attempts to demonstrate this interaction have thus far been unsuccessful. A recently developed bacterial two-hybrid protein interaction detection system was used to show that the N-terminal region of MR interacts with the cytoplasmic face of MerT. We also show that the cysteine residues on the cytoplasmic face of the MerT protein are required for maximal mercuric ion transport but not for the interaction with mercuric reductase.