Limits...
The PHA Depolymerase Engineering Database: A systematic analysis tool for the diverse family of polyhydroxyalkanoate (PHA) depolymerases.

Knoll M, Hamm TM, Wagner F, Martinez V, Pleiss J - BMC Bioinformatics (2009)

Bottom Line: Polyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra- or extracellular PHA depolymerases.PHA depolymerases are very diverse in sequence and substrate specificity, but share a common alpha/beta-hydrolase fold and a catalytic triad, which is also found in other alpha/beta-hydrolases.The DED is a valuable tool which can be applied to identify new PHA depolymerase sequences from complete genomes in silico, to classify PHA depolymerases, to predict their biochemical properties, and to design enzyme variants with improved properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring, Germany. michael.knoll@itb.uni-stuttgart.de

ABSTRACT

Background: Polyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra- or extracellular PHA depolymerases. PHA depolymerases are very diverse in sequence and substrate specificity, but share a common alpha/beta-hydrolase fold and a catalytic triad, which is also found in other alpha/beta-hydrolases.

Results: The PHA Depolymerase Engineering Database (DED, http://www.ded.uni-stuttgart.de) has been established as a tool for systematic analysis of this enzyme family. The DED contains sequence entries of 587 PHA depolymerases, which were assigned to 8 superfamilies and 38 homologous families based on their sequence similarity. For each family, multiple sequence alignments and profile hidden Markov models are provided, and functionally relevant residues are annotated.

Conclusion: The DED is a valuable tool which can be applied to identify new PHA depolymerase sequences from complete genomes in silico, to classify PHA depolymerases, to predict their biochemical properties, and to design enzyme variants with improved properties.

Show MeSH
Top view of the binding site of the PHB Depolymerase from Penicillium funiculosum (PDB entry 2D80, [46]). The catalytic residues are marked in red, the hydrophobic residue at position x1 of the Gx1Sx2G motif is marked in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2666664&req=5

Figure 2: Top view of the binding site of the PHB Depolymerase from Penicillium funiculosum (PDB entry 2D80, [46]). The catalytic residues are marked in red, the hydrophobic residue at position x1 of the Gx1Sx2G motif is marked in blue.

Mentions: All PHA depolymerases in the DED possess a lipase box around the catalytic serine with a Gx1Sx2G sequence motif with the exception of the family of intracellular nPHASCL depolymerases (no lipase box), which possess a catalytic cysteine instead of the lipase box. For particular PHA depolymerases it has been previously described that a hydrophobic residue is found at position x1 within the Gx1Sx2G motif [4,9,45]. This seems to be a common feature of almost all PHA depolymerases as seen from a systematic analysis of the DED family multiple sequence alignments. Thus, compared to other α/β-hydrolases like lipases and esterases, where a polar residue is most frequently found at position x1, this conserved residue of the Gx1Sx2G motif might be relevant to differentiate between lipases or esterases and PHA depolymerases on sequence level. This hydrophobic residue is solvent exposed and located near the catalytic serine at the bottom of a deep cleft, as seen in the structure of the PHB depolymerase from Penicillium funiculosum (PDB entry 2D80) [46] (Fig. 2). The hydrophobic residue at position x1 is tryptophan and isoleucine for the families of intracellular nPHASCL depolymerases (lipase box) and periplasmatic PHA depolymerases, respectively. For the family of intracellular nPHAMCL depolymerases, the residue at position x1 is valine for almost all proteins. Although not possessing a lipase box, but utilizing a catalytic cysteine, all family members of the family of intracellular nPHASCL depolymerases (no lipase box) also have a hydrophobic residue (almost all valine) at position cysteine-1. While the hydrophobic residue at position x1 differs among the families of intracellular PHA depolymerases, leucine and isoleucine are the most frequent residues at this position for extracellular PHA depolymerases. While all proteins of the family of extracellular dPHASCL depolymerases (catalytic domain type 2) possess a hydrophobic residue at position x1, only 81% of the proteins of the family of extracellular dPHASCL depolymerases (catalytic domain type 1) have a hydrophobic residue at position x1. All extracellular dPHAMCL depolymerases have an isoleucine at position x1. One exception is the family of extracellular nPHASCL depolymerases, which neither possess a typical Gx1Sx2G motif nor has a hydrophobic residue a position x1. In this family, the Gx1Sx2G motif is altered to a AHSMG motif which can also be found in the family of Bacillus lipases (homologous family abH18.01 in the LED, ). One family member of this special family is the PHB depolymerase from Paucimonas lemoignei, for which also structure information is available (PDB entry: 2VTV) [6,47]. This PHB depolymerase has also special biochemical properties, as it is an extracellular nPHASCL depolymerase degrading native granules, and is the only experimentally validated extracellular PHASCL depolymerase not having a substrate binding domain. Within lipases and esterases, a polar residue is typically found at position x1. However, a few exception also exist among lipases and esterases, such as the of Candida antarctica lipase like family (homologous family abH37 in the LED) and the family of Bacillus carboxylesterases (abH11.1).


The PHA Depolymerase Engineering Database: A systematic analysis tool for the diverse family of polyhydroxyalkanoate (PHA) depolymerases.

Knoll M, Hamm TM, Wagner F, Martinez V, Pleiss J - BMC Bioinformatics (2009)

Top view of the binding site of the PHB Depolymerase from Penicillium funiculosum (PDB entry 2D80, [46]). The catalytic residues are marked in red, the hydrophobic residue at position x1 of the Gx1Sx2G motif is marked in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666664&req=5

Figure 2: Top view of the binding site of the PHB Depolymerase from Penicillium funiculosum (PDB entry 2D80, [46]). The catalytic residues are marked in red, the hydrophobic residue at position x1 of the Gx1Sx2G motif is marked in blue.
Mentions: All PHA depolymerases in the DED possess a lipase box around the catalytic serine with a Gx1Sx2G sequence motif with the exception of the family of intracellular nPHASCL depolymerases (no lipase box), which possess a catalytic cysteine instead of the lipase box. For particular PHA depolymerases it has been previously described that a hydrophobic residue is found at position x1 within the Gx1Sx2G motif [4,9,45]. This seems to be a common feature of almost all PHA depolymerases as seen from a systematic analysis of the DED family multiple sequence alignments. Thus, compared to other α/β-hydrolases like lipases and esterases, where a polar residue is most frequently found at position x1, this conserved residue of the Gx1Sx2G motif might be relevant to differentiate between lipases or esterases and PHA depolymerases on sequence level. This hydrophobic residue is solvent exposed and located near the catalytic serine at the bottom of a deep cleft, as seen in the structure of the PHB depolymerase from Penicillium funiculosum (PDB entry 2D80) [46] (Fig. 2). The hydrophobic residue at position x1 is tryptophan and isoleucine for the families of intracellular nPHASCL depolymerases (lipase box) and periplasmatic PHA depolymerases, respectively. For the family of intracellular nPHAMCL depolymerases, the residue at position x1 is valine for almost all proteins. Although not possessing a lipase box, but utilizing a catalytic cysteine, all family members of the family of intracellular nPHASCL depolymerases (no lipase box) also have a hydrophobic residue (almost all valine) at position cysteine-1. While the hydrophobic residue at position x1 differs among the families of intracellular PHA depolymerases, leucine and isoleucine are the most frequent residues at this position for extracellular PHA depolymerases. While all proteins of the family of extracellular dPHASCL depolymerases (catalytic domain type 2) possess a hydrophobic residue at position x1, only 81% of the proteins of the family of extracellular dPHASCL depolymerases (catalytic domain type 1) have a hydrophobic residue at position x1. All extracellular dPHAMCL depolymerases have an isoleucine at position x1. One exception is the family of extracellular nPHASCL depolymerases, which neither possess a typical Gx1Sx2G motif nor has a hydrophobic residue a position x1. In this family, the Gx1Sx2G motif is altered to a AHSMG motif which can also be found in the family of Bacillus lipases (homologous family abH18.01 in the LED, ). One family member of this special family is the PHB depolymerase from Paucimonas lemoignei, for which also structure information is available (PDB entry: 2VTV) [6,47]. This PHB depolymerase has also special biochemical properties, as it is an extracellular nPHASCL depolymerase degrading native granules, and is the only experimentally validated extracellular PHASCL depolymerase not having a substrate binding domain. Within lipases and esterases, a polar residue is typically found at position x1. However, a few exception also exist among lipases and esterases, such as the of Candida antarctica lipase like family (homologous family abH37 in the LED) and the family of Bacillus carboxylesterases (abH11.1).

Bottom Line: Polyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra- or extracellular PHA depolymerases.PHA depolymerases are very diverse in sequence and substrate specificity, but share a common alpha/beta-hydrolase fold and a catalytic triad, which is also found in other alpha/beta-hydrolases.The DED is a valuable tool which can be applied to identify new PHA depolymerase sequences from complete genomes in silico, to classify PHA depolymerases, to predict their biochemical properties, and to design enzyme variants with improved properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring, Germany. michael.knoll@itb.uni-stuttgart.de

ABSTRACT

Background: Polyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra- or extracellular PHA depolymerases. PHA depolymerases are very diverse in sequence and substrate specificity, but share a common alpha/beta-hydrolase fold and a catalytic triad, which is also found in other alpha/beta-hydrolases.

Results: The PHA Depolymerase Engineering Database (DED, http://www.ded.uni-stuttgart.de) has been established as a tool for systematic analysis of this enzyme family. The DED contains sequence entries of 587 PHA depolymerases, which were assigned to 8 superfamilies and 38 homologous families based on their sequence similarity. For each family, multiple sequence alignments and profile hidden Markov models are provided, and functionally relevant residues are annotated.

Conclusion: The DED is a valuable tool which can be applied to identify new PHA depolymerase sequences from complete genomes in silico, to classify PHA depolymerases, to predict their biochemical properties, and to design enzyme variants with improved properties.

Show MeSH