A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholatecoated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-␣-benzoyl-L-arginine-4-nitranilide, or starch. The purified enzyme (M r 36,209) was remarkably stable and active at high temperature (60°C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested.Poly((R)-3-hydroxyalkanoic acids) (PHAs) 1 are a class of storage compounds that are synthesized during unbalanced growth by many bacteria. PHAs are deposited intracellularly in the form of inclusion bodies ("granules") to levels up to 90% of the cellular dry weight. The subject was reviewed recently (1). Poly((R)-3-hydroxybutyric acid) (PHB) is the most abundant polyester in bacteria. Bacterial copolymers containing randomly distributed (R)-3-hydroxybutyric and (R)-3-hydroxyvaleric units (poly(3HB-co-3HV)) have been commercialized for over a decade under the trade name Biopol ® . Any research on the biodegradation of PHA should clearly distinguish between (i) extracellular PHA degradation and (ii) intracellular PHA degradation. (i) Extracellular degradation is the utilization of an exogenous carbon/energy source by a notnecessarily-accumulating microorganism. The source of this extracellular polymer is PHA-released by accumulating cells after death. The ability to degrade PHA is widely distributed among bacteria and depends on the secretion of specific PHA depolymerases that are carboxyesterases (EC 3.1.1) a...
Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.The mechanism of degradation of denatured, exogenous, crystalline poly(3-hydroxybutyrate) (PHB) by extracellular PHB depolymerases has been extensively studied during the last decade (for a recent review, see reference 6). However, intracellular PHB (i-PHB) degradation, i. e., the mobilization of previously accumulated amorphous PHB, is poorly understood. The beneficial effect of accumulated PHB on survival in the absence of a carbon source has been described for several species, including Ralstonia eutropha (4), Legionella pneumophila (5), and Hydrogenophaga pseudoflava (15). Recently, a DNA sequence of a putative i-PHB depolymerase of R. eutropha H16 was determined (GenBank accession no. AB017612). However, the physiological relevance of this gene during mobilization of PHB has not been investigated.PHB-rich cells of R. eutropha H16 (DSM428) were resuspended and incubated in carbon-free mineral-salt medium (12) with (mobilization) or without (control) NH 4 Cl for 6 days. The number of viable cells, in CFU per milliliter, increased about fourfold in the presence of NH 4 Cl within 30 h and remained constantly high for 6 days ( Fig. 1). In contrast, the CFU/ml remained almost unchanged in the absence of a nitrogen source. The PHB content of strain H16 (assayed through gas chromatography [1]) rapidly decreased from 70 to Ϸ30% during the first day of mobilization and decreased further, below 20%, after 6 days (Fig. 1). The PHB content of the control decreased only very little. The CFU/ml of the PHB-free mutant PHB Ϫ 4 (DSM541) decreased rapidly by several orders of magnitude after 3 days regardless of the absence or presence of a nitrogen source. Similar results were obtained with other bacteria such as Acidovorax delafieldii (DSM50403), Alcaligenes faecalis (14), a Comamonas sp. (DSM6781) and two Paracoccus denitrificans strains (DSM1404 and DSM413); however, the amount of PHB accumulation and the increase of the CFU/ml during mobilization of PHB were not as pronounced for these bacteria as for R. eutropha (data not shown). We conclude that R. eutropha H16 and other bacteria are able to mobilize previously accumulated PHB and to use the degradation products for one or two cell divisions even in the absence of an exogenous carbon source. Very little PHB is mobilized if ...
Poly(3-hydroxybutyrate) (PHB) is a compound that stores carbon and energy in many bacteria and can account for up to 90% of the cellular dry weight during unbalanced growth (for recent reviews see references 3 and 20). Large quantities of PHB can be isolated by solvent extraction, and due to its thermoplastic properties and biodegradation to water and carbon dioxide PHB has attracted academic and industrial interest over the past two decades. This polymer has been commercialized under the trade name BIOPOL.Accumulated PHB can be hydrolyzed by the accumulating strain itself during periods of starvation (intracellular PHB hydrolysis by intracellular PHB depolymerases) or by other microorganisms after release of the polymer from the accumulating strain (extracellular PHB hydrolysis by extracellular PHB depolymerases). The differentiation between extra-and intracellular degradation is necessary because PHB can be present in two biophysical conformations. In vivo, the polymer is completely amorphous (native) and is covered by a surface layer that is about one-half the size of a cytoplasmic membrane (1) and consists of proteins (so-called phasins) and phospholipids (6, 22, 34, 38). In Ralstonia eutropha H16 the major phasin protein is PhaP, which is involved in synthesis, morphology, and regulation of PHB synthesis (12,27,37,39,41,42). After release of the polymer from the cell (e.g., after cell lysis or solvent extraction) or after removal or damage of the surface layer, the polymer denatures and becomes paracrystalline. For the sake of clarity PHB in its intact intracellular, amorphous form is called native PHB (nPHB), and extracellular, partially crystalline PHB without a surface layer or with a damaged surface layer is called denatured PHB (dPHB). Most enzymes that hydrolyze PHB are specific for one of the two forms (nPHB or dPHB). For example, extracellular PHB depolymerases that are released from PHB-degrading bacteria so that PHB can be used as an exogenous carbon source are able to hydrolyze dPHB. Intracellular PHB depolymerases are necessary for utilization of the previously accumulated PHB by
The substrate specificity of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivoransand its corrinoid cofactor were studied. Besides reduced methyl viologen, titanium(III) citrate could serve as electron donor for reductive dehalogenation of tetrachloroethene (PCE) and trichloroethene to cis-1,2-dichloroethene. In addition to chlorinated ethenes, chlorinated propenes were reductively dechlorinated solely by the native enzyme. trans-1,3-Dichloropropene, 1,1,3-trichloropropene and 2,3-dichloropropene were reduced to a mixture of mono-chloropropenes, 1,1-dichloropropene, and 2-chloropropene, respectively. Other halogenated compounds that were rapidly reduced by the enzyme were also dehalogenated abiotically by the heat-inactivated enzyme and by commercially available cyanocobalamin. The rate of this abiotic reaction was dependent on the number and type of halogen substituents and on the type of catalyst. The corrinoid cofactor purified from the tetrachloroethene dehalogenase of D. multivorans exhibited an activity about 50-fold higher than that of cyanocobalamin (vitamin B(12)) with trichloroacetate as electron acceptor, indicating that the corrinoid cofactor of the PCE dehalogenase is not cyanocobalamin. Corrinoids catalyzed the rapid dehalogenation of trichloroacetic acid. The rate was proportional to the amount of, e.g. cyanocobalamin; therefore, the reductive dehalogenation assay can be used for the sensitive and rapid quantification of this cofactor.
Xanthomonas sp. secretes an extracellular protein (M r W70 þ 5 kDa) during growth on purified natural rubber [poly(1,4-cis-isoprene)] but not during growth on water-soluble carbon sources such as glucose or gluconate. A 1.3 kbp DNA fragment coding for an internal part of the structural gene of the 70 kDa protein was amplified by nested polymerase chain reaction (PCR) using amino acid sequence information obtained after Edman degradation of selected trypsin-generated peptides of the purified 70 kDa protein. The PCR product was used as a DNA probe to clone the complete structural gene from genomic DNA of Xanthomonas sp. The sequenced DNA contained a 2037 bp open reading frame which coded for a polypeptide of 678 amino acids (M r 74.6 kDa) and which included the features of the N-terminal signal peptidase cleavage site (M r W72.9 kDa for the mature protein). Analysis of the amino acid sequence revealed the presence of two heme binding motifs (CXXCH) and a W20 amino acids long sequence that is conserved in the Paracoccus denitrificans and Pseudomonas aeruginosa diheme cytochrome c peroxidases (CCPs). This region includes a histidine residue (H 519 in Xanthomonas sp. and H 265 and H 271 in the Pseudomonas strains, respectively) that is essential for activity in CCPs and that is also conserved in other bacterial oxidases. Blast analysis confirmed the relatedness of the 70 kDa protein to heme-containing oxidases and suggested that it is a member of a new family of relatively large (W500 to W1000 amino acids) extracellular proteins with so far unknown function being only far related in amino acid sequence to P. denitrificans and P. aeruginosa CCPs.
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