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Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory.

Satomura A, Kuroda K, Ueda M - PLoS ONE (2015)

Bottom Line: In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide.The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate.These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan; Japan Society for the Promotion of Science, Sakyo-ku, Kyoto, Japan.

ABSTRACT
Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROLimp was more stable than mROLWT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity.

No MeSH data available.


Structure of mROLWT, modeled using the SWISS-MODEL program.The structure of mROLWT was modeled based on the open-lid structure of Rhizomucor miehei lipase (Protein Data Bank [PDB] ID: 4TGL), and visualized using PyMOL. The active site residues, S242, D301, and H354, are colored orange. The magenta-colored α-helix represents the lid domain. Green residues indicate hydrophilic amino acids and white residues indicate hydrophobic amino acids.
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pone.0124545.g005: Structure of mROLWT, modeled using the SWISS-MODEL program.The structure of mROLWT was modeled based on the open-lid structure of Rhizomucor miehei lipase (Protein Data Bank [PDB] ID: 4TGL), and visualized using PyMOL. The active site residues, S242, D301, and H354, are colored orange. The magenta-colored α-helix represents the lid domain. Green residues indicate hydrophilic amino acids and white residues indicate hydrophobic amino acids.

Mentions: Most lipases have a lid domain that conceals the active site [16, 17]. In the case of Rhizomucor miehei lipase, which has 50% sequence homology to ROL, an open lid that forms a hydrophobic groove is important for substrate recognition [16]. The tertiary structure of mROLWT, modeled from the open-lid structure of R. miehei lipase, revealed that the open lid of mROLWT did not form a hydrophobic groove around the active site (Fig 5). The mutated propeptide probably affected the conformation of the lid domain and allowed the open lid to form a hydrophobic groove to interact with substrates composed of medium-chain length acyl groups and a hydrophobic peptidase-specific substrate. A previous study showed that mutations in the lid domain changed the substrate specificity of mROL [17], indicating that the open lid plays a critical role in substrate recognition. Because the lid domain and surrounding residues in ROL contain hydrophilic residues such as Arg184, Ser185, Thr188, and Asp189 (Fig 1), the additional hydrophilic residues introduced in the mutated propeptide might interact with the residues of the lid domain via hydrogen bonding. Interestingly, another propeptide mutant in which residues Gln50–Glu57 (which are essential for folding) were replaced with eight hydrophilic Asn residues also exhibited “protein folding memory” and induced peptidase activity in the mature domain of ROL (named mROLimp2; Fig 4A and 4B). These results strongly suggest that the hydrophilic residues in the propeptide interacted with the lid domain and surrounding residues in a different manner compared with wild type propeptide. CD spectrum analyses showed change in the secondary structure and thermal stability of mROLimp; however, it did not reveal detailed folding mechanisms of mROLWT and mROLimp. Crystallization analyses of mROLWT and mROLimp will provide further insight into the phenomenon of “protein folding memory”. Although we have attempted to crystallize ROL according to the crystallization methods for homologous lipases such as R. niveus lipase and R. miehei lipase, but it has been difficult. The elaborated structural characterizations of mROLs would be our subject of the future work.


Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory.

Satomura A, Kuroda K, Ueda M - PLoS ONE (2015)

Structure of mROLWT, modeled using the SWISS-MODEL program.The structure of mROLWT was modeled based on the open-lid structure of Rhizomucor miehei lipase (Protein Data Bank [PDB] ID: 4TGL), and visualized using PyMOL. The active site residues, S242, D301, and H354, are colored orange. The magenta-colored α-helix represents the lid domain. Green residues indicate hydrophilic amino acids and white residues indicate hydrophobic amino acids.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430139&req=5

pone.0124545.g005: Structure of mROLWT, modeled using the SWISS-MODEL program.The structure of mROLWT was modeled based on the open-lid structure of Rhizomucor miehei lipase (Protein Data Bank [PDB] ID: 4TGL), and visualized using PyMOL. The active site residues, S242, D301, and H354, are colored orange. The magenta-colored α-helix represents the lid domain. Green residues indicate hydrophilic amino acids and white residues indicate hydrophobic amino acids.
Mentions: Most lipases have a lid domain that conceals the active site [16, 17]. In the case of Rhizomucor miehei lipase, which has 50% sequence homology to ROL, an open lid that forms a hydrophobic groove is important for substrate recognition [16]. The tertiary structure of mROLWT, modeled from the open-lid structure of R. miehei lipase, revealed that the open lid of mROLWT did not form a hydrophobic groove around the active site (Fig 5). The mutated propeptide probably affected the conformation of the lid domain and allowed the open lid to form a hydrophobic groove to interact with substrates composed of medium-chain length acyl groups and a hydrophobic peptidase-specific substrate. A previous study showed that mutations in the lid domain changed the substrate specificity of mROL [17], indicating that the open lid plays a critical role in substrate recognition. Because the lid domain and surrounding residues in ROL contain hydrophilic residues such as Arg184, Ser185, Thr188, and Asp189 (Fig 1), the additional hydrophilic residues introduced in the mutated propeptide might interact with the residues of the lid domain via hydrogen bonding. Interestingly, another propeptide mutant in which residues Gln50–Glu57 (which are essential for folding) were replaced with eight hydrophilic Asn residues also exhibited “protein folding memory” and induced peptidase activity in the mature domain of ROL (named mROLimp2; Fig 4A and 4B). These results strongly suggest that the hydrophilic residues in the propeptide interacted with the lid domain and surrounding residues in a different manner compared with wild type propeptide. CD spectrum analyses showed change in the secondary structure and thermal stability of mROLimp; however, it did not reveal detailed folding mechanisms of mROLWT and mROLimp. Crystallization analyses of mROLWT and mROLimp will provide further insight into the phenomenon of “protein folding memory”. Although we have attempted to crystallize ROL according to the crystallization methods for homologous lipases such as R. niveus lipase and R. miehei lipase, but it has been difficult. The elaborated structural characterizations of mROLs would be our subject of the future work.

Bottom Line: In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide.The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate.These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan; Japan Society for the Promotion of Science, Sakyo-ku, Kyoto, Japan.

ABSTRACT
Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROLimp was more stable than mROLWT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity.

No MeSH data available.