Lipase Box Research Articles

Enzymatic hydrolysis of chemosynthesized atactic poly(3-hydroxybutyrate) by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4 and Ralstonia pickettii T1.

The enzymatic degradability of chemosynthesized atactic poly([R,S]-3-hydroxybutyrate) [a-P(3HB)] by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pickettii T1 (PhaZ(ral)) and Acidovorax Sp. TP4 (PhaZ(aci)), defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were studied. The enzymatic degradation of a-P(3HB) by PhaZ(aci) depolymerase was confirmed from the results of weight loss and the scanning electron micrographs. The degradation products were characterized by one- and two-dimension (1)H NMR spectroscopy. It was found that a-P(3HB) could be degraded into monomer, dimer, and trimer by PhaZ(aci) depolymerase at temperatures ranging from 4 to 20 degrees C, while a-P(3HB) could hardly be hydrolyzed by PhaZ(ral) depolymerase in the same temperature range. These results suggested that the chemosynthesized a-P(3HB) could be degraded in the pure state by natural PHA depolymerase.

Cloning of an intracellular Poly[D(-)-3-Hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product.

An intracellular poly[D(-)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(-)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.

Properties of a Poly(3-hydroxybutyrate) Depolymerase from Penicillium funiculosum

A poly(3-hydroxybutyrate) (PHB) depolymerase was purified from a fungus, Penicillium funiculosum (IFO6345), with phenyl-Toyopearl and its properties were compared with those of other PHB depolymerases. The molecular mass of the purified enzyme was estimated at about 33 kDa by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The pH optimum and pI were 6.5 and 6.5, respectively. The purified protein showed affinity to Con A-Sepharose, indicating that it is a glycoprotein. Diisopropylfluorophosphate and dithiothreitol inhibited the depolymerase activity completely. The N-terminal amino acid sequence of the purified enzyme was TALPAFNVNPNSVSVSGLSSGGYMAAQL, which contained a “lipase box” sequence. This purified enzyme is one of the extracellular PHB depolymerase which belong to serine esterase. The purified enzyme showed relatively strong hydrolytic activity against 3-hydroxybutyrate oligomers compared with its PHB-degrading activity. PHB-binding experiments showed that P. funiculosum depolymerase has the weakest affinity for PHB of all the depolymerases examined.

Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases

The substrate specificities of three extracellular polyhydroxybutyrate (PHB) depolymerases from Alcaligenes faecalis (PhaZ Afa), Pseudomonas stutzeri (PhaZ Pst), and Comamonas acidovorans (PhaZ Cac), which are grouped into types A and B based on the position of a lipase box sequence in the catalytic domain, were examined for films of 12 different aliphatic polyesters. Each of these PHB depolymerases used was capable of hydrolyzing poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxypropionate) (P(3HP)), poly(4-hydroxybutyrate) (P(4HB)), poly(ethylene succinate) (PESU), and poly(ethylene adipate) (PEA) but could not hydrolyze another seven polyesters. In addition, the binding characteristics of substrate binding domains from PhaZ Afa, PhaZ Cac, and PHB depolymerase from Comamonas testosteroni (PhaZ Cte) were studied by using fusions with glutathione S-transferase (GST). All of fusion proteins adsorbed strongly on the surfaces of polyester granules of P(3HB), P(3HP), and poly(2-hydroxypropionate) (P(2HP)) which was not hydrolyzed by the PHB depolymerases used in this study, while they did not bind on Avicel and chitin granules. The adsorption kinetics of the fusion proteins to the surface of P(3HB) and P(2HP) granules were found to obey the Langmuir isotherm. The cross-area per molecule of fusion protein bound to P(3HB) granules was estimated to be 12±4 nm 2/molecule. It has been suggested that the active sites in catalytic domains of PHB depolymerases have a similar conformational structure, and that several amino acids in substrate-binding domains of PHB depolymerases interact specifically with the surface of polyesters.

Biochemical and Genetic Characterization of an Extracellular Poly(3-Hydroxybutyrate) Depolymerase from Acidovorax Sp. Strain TP4

To determine the properties of enzymes from bacteria that degrade polypropiolactone (PPL), we isolated 13 PPL-degrading bacteria from pond water, river water, and soil. Nine of these strains were identified as Acidovorax sp., three as Variovorax paradoxus, and one as Sphingomonas paucimobilis. All the isolates also degraded poly(3-hydroxybutyrate) (PHB). A PPL-degrading enzyme was purified to electrophoretical homogeneity from one of these bacteria, designated Acidovorax sp. TP4. The purified enzyme also degraded PHB. The molecular weight of the enzyme was estimated as about 50,000. The enzyme activity was inhibited by diisopropylfluorophosphate, dithiothreitol, and Triton X-100. The structural gene of the depolymerase was cloned in Escherichia coli. The nucleotide sequence of the cloned DNA fragment contained an open reading frame (1476 bp) specifying a protein with a deduced molecular weight of 50,961 (491 amino acids). The deduced overall sequence was very similar to that of a PHB depolymerase of Comamonas acidovorans YM1609. From these results it was concluded that the isolated PPL-degrading enzyme belongs to the class of PHB depolymerases. A conserved amino acid sequence, Gly-X1-Ser-X2-Gly (lipase box), was found at the N-terminal side of the amino acid sequence. Site-directed mutagenesis of the TP4 enzyme confirmed that 20Ser in the lipase box was essential for the enzyme activity. This is the first report of the isolation a PHB depolymerase from Acidovorax.

Enzymatic Degradation and Adsorption on Poly[(R)-3-hydroxybutyrate] Single Crystals with Two Types of Extracellular PHB Depolymerases from Comamonas acidovorans YM1609 and Alcaligenes faecalis T1

Poly[(R)-3-hydroxybutyrate] (P(3HB)) single crystals, which had different morphologies with and without chain-folding surfaces, were grown from dilute solutions of chloroform and ethanol. Two types of extracellular PHB depolymerases purified from Alcaligenes faecalis T1 and Comamonas acidovorans YM1609, defined as types I and II according to the position of the lipase box in the catalytic domain, were used in the enzymatic degradation and adsorption experiments. The enzymatic degradation of P(3HB) single crystals was investigated by means of transmission electron microscopy, atomic force microscopy, and gel-permeation chromatography. Adsorption of PHB depolymerase on P(3HB) single crystals was examined using an immuno-gold labeling technique. Enzymatic degradation of single crystals progressed from the edges and ends of crystals to yield the narrow cracks and the small crystal fragments along their long axis, independent of both surface morphologies of single crystals and types of extracellular PHB depolymerases. Lamellar thicknesses of single crystals and molecular weights of P(3HB) chains remained unchanged during the enzymatic hydrolysis. Adsorption of extracellular PHB depolymerase demonstrated a homogeneous distribution of enzyme molecules on the chain-folding surface of crystals. The above results suggest that the adsorption of enzyme increases with the mobility of P(3HB) chains of single crystals as a whole and that the endo−exo type attack by the active-site of PHB depolymerase takes place preferentially at the disordered chain-packing regions of crystal edges and ends rather than the chain-folding surfaces of single crystals, in spite of the difference in the structure of enzymes.

Determination of the active sites serine of the poly (3-hydroxybutyrate) depolymerases ofPseudomonas lemoignei(PhaZ5) and ofAlcaligenes faecalis

Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S138 (P. lemoignei) and S139 (A. faecalis) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase box of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S195 (P. lemoignei) and S196 (A. faecalis), resulted in mutant proteins with almost full activity and proved that S195 and S196 are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.

Sequence of the bphD gene encoding 2-hydroxy-6-oxo-(phenyl/ chlorophenyl)hexa-2,4-dienoic acid (HOP/cPDA) hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway in Comamonas testosteroni: Evidence suggesting involvement of Ser 112 in catalytic activity

The nucleotide sequence of bphD, encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway of Comamonas testosteroni strain B-356, was determined. Comparison of the deduced amino-acid sequence with published sequences led to the identification of a ‘lipase box’, containing a consensus pentapeptide sequence GlyXaaSerXaaGly. This suggested that the mechanism of action of this enzyme may involve an Asp-Ser-His catalytic triad similar to that of classical lipases and serine hydrolases. Further biochemical and genetic evidence for the active-site involvement of Ser 112 was obtained by showing that a semipurified enzyme was inhibited by PMSF, a classic inhibitor of serine hydrolases, and by site-directed Ser 112→Ala mutagenesis.