Gene_locus Report for: psele-PHAZ7

Poly-(R)-hydroxyalkanoates (PHAs) are bacterial polyesters that are degraded by a group of enzymes known as PHA depolymerases. Paucimonas lemoignei PhaZ7 depolymerase is the only extracellular depolymerase that has been described as being active towards amorphous PHAs. A previously determined crystal structure of PhaZ7 revealed an alpha/beta-hydrolase fold and a Ser-His-Asp catalytic triad. In order to address questions regarding the catalytic mechanism and substrate binding, the atomic resolution structure of PhaZ7 was determined after cocrystallization with the protease inhibitor PMSF. The reported structure has the highest resolution (1.2 A) of currently known depolymerase structures and shows a sulfur dioxide molecule covalently attached to the active-site residue Ser136. Structural comparison with the free PhaZ7 structure (1.45 A resolution) revealed no major changes in the active site, suggesting a preformed catalytic triad. The oxyanion hole was found to be formed by the amide groups of Met137 and Asn49. Nine well ordered water molecules were located in the active site. Manual docking of a substrate trimer showed that the positions of these water molecules coincide well with the substrate atoms. It is proposed that these water molecules are displaced upon binding of the substrate. Furthermore, conformational changes were identified after comparison with a previously determined PhaZ7 dimer structure in a different space group. The changes were located in surface loops involved in dimer formation, indicating some flexibility of these loops and their possible involvement in polyester binding.

The crystal structure of poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 purified from Paucimonas lemoignei was determined at 1.90 A resolution. The structure consists of a single domain with an alpha/beta hydrolase fold in its core. The active site is analogous to that of serine esterases/lipases and is characterized by the presence of a catalytic triad comprising Ser136, Asp242, and His306. Comparison with other structures in the Protein Data Bank showed a high level of similarity with the Bacillus subtilis lipase LipA (RMSD, 1.55 A). Structural comparison with Penicillium funiculosum PHB depolymerase, the only PHB depolymerase whose structure is already known, revealed significant differences, resulting in an RMSD of 2.80-3.58 A. The two enzymes appear to utilize different types of solvent-exposed residues for biopolymer binding, with aliphatic and hydroxyl residues used in P. funiculosum PHB depolymerase and aromatic residues in PhaZ7. Moreover, the active site of P. funiculosum PHB depolymerase is accessible to the substrate in contrast to the active site of PhaZ7, which is buried. Hence, considerable conformational changes are required in PhaZ7 for the creation of a channel leading to the active site. Taken together, the structural data suggest that PhaZ7 and P. funiculosum PHB depolymerase have adopted different strategies for effective substrate binding in response to their diverse substrate specificity and the lack of a substrate-binding domain.

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 cholate-coated 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-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees 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.

An open and a closed conformation of a surface loop in PhaZ7 extracellular poly(3-hydroxybutyrate) depolymerase were identified in two high-resolution crystal structures of a PhaZ7 Y105E mutant. Molecular dynamics (MD) simulations revealed high root mean square fluctuations (RMSF) of the 281-295 loop, in particular at residue Asp289 (RMSF 7.62 A). Covalent docking between a 3-hydroxybutyric acid trimer and the catalytic residue Ser136 showed that the binding energy of the substrate is significantly more favorable in the open loop conformation compared to that in the closed loop conformation. MD simulations with the substrate covalently bound depicted 1 A RMSF higher values for the residues 281-295 in comparison to the apo (substrate-free) form. In addition, the presence of the substrate in the active site enhanced the ability of the loop to adopt a closed form. Taken together, the analysis suggests that the flexible loop 281-295 of PhaZ7 depolymerase can act as a lid domain to control substrate access to the active site of the enzyme. Proteins 2017; 85:1351-1361. (c) 2017 Wiley Periodicals, Inc.

Five amino acids (Y105, Y176, Y189, Y189, W207) that constitute the substrate binding site of PHB depolymerase PhaZ7 were identified. All residues are located at a single surface-exposed location of PhaZ7. Exchange of these amino acids by less hydrophobic, hydrophilic or negatively charged residues reduced binding of PhaZ7 to PHB. Modifications of other residues at the PhaZ7 surface (F9, Y66, Y103, Y124, Y169, Y172, Y173, F198, Y203, Y204, F251, W252) had no effect on substrate binding. The PhaZ7 wild-type protein, three muteins with single amino acid exchanges (Y105A, Y105E, Y190E), a PhaZ7 variant with deletion of residues 202-208, and PhaZ7 in which the active-site serine had been replaced by alanine (S136A) were crystallized and their structures were determined at 1.6-2.0 A resolution. The structures were almost identical but revealed flexibility of some regions. Structural analysis of PhaZ7 (S136A) with bound 3-hydroxybutyrate tetramer showed that the substrate binds in a cleft that is composed of Y105, Y176, Y189 and Y190 and thus confirmed the data obtained by site-directed mutagenesis. To the best of our knowledge this is the first example in which the substrate binding site of a PHB depolymerase is documented at a molecular and structural level.

Poly-(R)-hydroxyalkanoates (PHAs) are bacterial polyesters that are degraded by a group of enzymes known as PHA depolymerases. Paucimonas lemoignei PhaZ7 depolymerase is the only extracellular depolymerase that has been described as being active towards amorphous PHAs. A previously determined crystal structure of PhaZ7 revealed an alpha/beta-hydrolase fold and a Ser-His-Asp catalytic triad. In order to address questions regarding the catalytic mechanism and substrate binding, the atomic resolution structure of PhaZ7 was determined after cocrystallization with the protease inhibitor PMSF. The reported structure has the highest resolution (1.2 A) of currently known depolymerase structures and shows a sulfur dioxide molecule covalently attached to the active-site residue Ser136. Structural comparison with the free PhaZ7 structure (1.45 A resolution) revealed no major changes in the active site, suggesting a preformed catalytic triad. The oxyanion hole was found to be formed by the amide groups of Met137 and Asn49. Nine well ordered water molecules were located in the active site. Manual docking of a substrate trimer showed that the positions of these water molecules coincide well with the substrate atoms. It is proposed that these water molecules are displaced upon binding of the substrate. Furthermore, conformational changes were identified after comparison with a previously determined PhaZ7 dimer structure in a different space group. The changes were located in surface loops involved in dimer formation, indicating some flexibility of these loops and their possible involvement in polyester binding.

The crystal structure of poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 purified from Paucimonas lemoignei was determined at 1.90 A resolution. The structure consists of a single domain with an alpha/beta hydrolase fold in its core. The active site is analogous to that of serine esterases/lipases and is characterized by the presence of a catalytic triad comprising Ser136, Asp242, and His306. Comparison with other structures in the Protein Data Bank showed a high level of similarity with the Bacillus subtilis lipase LipA (RMSD, 1.55 A). Structural comparison with Penicillium funiculosum PHB depolymerase, the only PHB depolymerase whose structure is already known, revealed significant differences, resulting in an RMSD of 2.80-3.58 A. The two enzymes appear to utilize different types of solvent-exposed residues for biopolymer binding, with aliphatic and hydroxyl residues used in P. funiculosum PHB depolymerase and aromatic residues in PhaZ7. Moreover, the active site of P. funiculosum PHB depolymerase is accessible to the substrate in contrast to the active site of PhaZ7, which is buried. Hence, considerable conformational changes are required in PhaZ7 for the creation of a channel leading to the active site. Taken together, the structural data suggest that PhaZ7 and P. funiculosum PHB depolymerase have adopted different strategies for effective substrate binding in response to their diverse substrate specificity and the lack of a substrate-binding domain.

Polyhydroxyalkanoates (PHA) are biodegradable polyesters that have attracted commercial and academic interest as environmentally friendly materials. A number of enzymes are able to degrade polyhydroxyalkanoates to water-soluble products. PhaZ7 poly(3-hydroxybutyrate) (PHB) depolymerase (EC 3.1.1.75), a 342-amino-acid hydrolase from the PHA-degrading bacterium Paucimonas lemoignei, has been found to possess substrate specificity for amorphous PHA. PhaZ7 was crystallized by the microdialysis method. Thin rod-like crystals were grown in low ionic strength solution and found to belong to the monoclinic space group C2, with unit-cell parameters a = 225.8, b = 46.5, c = 171.3, beta = 128.9 degrees. A complete data set was collected to 2.75 A resolution at 100 K using synchrotron radiation.

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 cholate-coated 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-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees 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.