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Biochemical analysis and structure determination of P aucimonas lemoignei poly(3‐hydroxybutyrate) ( PHB ) depolymerase PhaZ 7 muteins reveal the PHB binding site and details of substrate–enzyme interactions
Author(s) -
Jendrossek Dieter,
Hermawan Siska,
Subedi Bishwa,
Papageorgiou Anastassios C.
Publication year - 2013
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12391
Subject(s) - tetramer , binding site , mutagenesis , serine , amino acid , substrate (aquarium) , biochemistry , alanine , site directed mutagenesis , stereochemistry , chemistry , biology , enzyme , mutant , ecology , gene
Summary Five amino acids ( Y 105, Y 176, Y 189, Y 189, W 207) that constitute the substrate binding site of PHB depolymerase PhaZ 7 were identified. All residues are located at a single surface‐exposed location of PhaZ 7. Exchange of these amino acids by less hydrophobic, hydrophilic or negatively charged residues reduced binding of PhaZ 7 to PHB . Modifications of other residues at the PhaZ 7 surface ( F 9, Y 66, Y 103, Y 124, Y 169, Y 172, Y 173, F 198, Y 203, Y 204, F 251, W 252) had no effect on substrate binding. The PhaZ 7 wild‐type protein, three muteins with single amino acid exchanges ( Y 105 A , Y 105 E , Y 190 E ), a PhaZ 7 variant with deletion of residues 202–208, and PhaZ 7 in which the active‐site serine had been replaced by alanine ( S 136 A ) were crystallized and their structures were determined at 1.6–2.0 Å resolution. The structures were almost identical but revealed flexibility of some regions. Structural analysis of PhaZ 7 ( S 136 A ) with bound 3‐hydroxybutyrate tetramer showed that the substrate binds in a cleft that is composed of Y 105, Y 176, Y 189 and Y 190 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.