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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.


Sequence alignment of Rhizopus oryzae lipase (ROL) and ROL-related lipases.The full-length primary sequences of ROL, R. niveus lipase (RNL), R. stolonifer lipase (RSL), and R. chinensis lipase (RCL) are presented. Multiple-sequence alignments were generated using the ClustalW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The underlined sequences in the propeptide of ROL (Ser20–Gly37 and Ser38–Glu57) indicate the regions that are essential for secretion and folding of mROL, respectively. The underlined sequences in the mature domain of ROL (Phe183–Asp189) indicate the lid domain. The shadowed region indicates residues that were replaced with hydrophilic amino acids (VDDDDK). In the original host, R. oryzae, the propeptide is also cleaved between the Ala97 and Ser98 residues [21]; however in P. pastoris and S. cerevisiae, the secondary cleavage has not been observed [14, 22]. Therefore, in this study, we defined the propeptide domain as the region between residues 1 and 69 and the mature domain as the region between residues 70 and 366 of ROL.
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pone.0124545.g001: Sequence alignment of Rhizopus oryzae lipase (ROL) and ROL-related lipases.The full-length primary sequences of ROL, R. niveus lipase (RNL), R. stolonifer lipase (RSL), and R. chinensis lipase (RCL) are presented. Multiple-sequence alignments were generated using the ClustalW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The underlined sequences in the propeptide of ROL (Ser20–Gly37 and Ser38–Glu57) indicate the regions that are essential for secretion and folding of mROL, respectively. The underlined sequences in the mature domain of ROL (Phe183–Asp189) indicate the lid domain. The shadowed region indicates residues that were replaced with hydrophilic amino acids (VDDDDK). In the original host, R. oryzae, the propeptide is also cleaved between the Ala97 and Ser98 residues [21]; however in P. pastoris and S. cerevisiae, the secondary cleavage has not been observed [14, 22]. Therefore, in this study, we defined the propeptide domain as the region between residues 1 and 69 and the mature domain as the region between residues 70 and 366 of ROL.

Mentions: Our previous study revealed that residues Ser38–Glu57 of proROL were essential for the folding of mROL [6]. This region is highly conserved among 4 homologous lipases (Fig 1), and contains three charged amino acids (Asp41, Glu45, and Lys51) and many hydrophilic residues, indicating that proROL interacts with mROL via electrostatic and hydrophilic interactions. In particular, residues Gln50–Trp55 are highly hydrophilic. We replaced these residues with a more hydrophilic and charged amino acid sequence (VDDDDK) to alter the chaperoning function of this region. The mutated propeptide was named proROL-mut1 and the mature ROL folded by proROL-mut1 was named mROLimp (imprinted ROL) to distinguish it from mROLWT (mROL folded by wild type proROL). The two distinct mROLWT and mROLimp with the same primary sequence were produced and purified from P. pastoris (S1 Fig) using a two-step purification protocol (see Materials and Method). No apparent decrease in molecular weight was observed after endoglycosidase H (EndoH) treatment, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that mROLWT and mROLimp were not N-glycosylated. To confirm that proROL and proROL-mut1 were cleaved at the same site and that the primary sequences of mROLWT and mROLimp were identical, the protein bands were excised and analyzed by N-terminal sequencing. The analysis confirmed that both sequences of mROLWT and mROLimp started with DDNLVGGMTLD, indicating that the propeptides were cleaved at the same site and that the primary sequences of mROLWT and mROLimp were the same.


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

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

Sequence alignment of Rhizopus oryzae lipase (ROL) and ROL-related lipases.The full-length primary sequences of ROL, R. niveus lipase (RNL), R. stolonifer lipase (RSL), and R. chinensis lipase (RCL) are presented. Multiple-sequence alignments were generated using the ClustalW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The underlined sequences in the propeptide of ROL (Ser20–Gly37 and Ser38–Glu57) indicate the regions that are essential for secretion and folding of mROL, respectively. The underlined sequences in the mature domain of ROL (Phe183–Asp189) indicate the lid domain. The shadowed region indicates residues that were replaced with hydrophilic amino acids (VDDDDK). In the original host, R. oryzae, the propeptide is also cleaved between the Ala97 and Ser98 residues [21]; however in P. pastoris and S. cerevisiae, the secondary cleavage has not been observed [14, 22]. Therefore, in this study, we defined the propeptide domain as the region between residues 1 and 69 and the mature domain as the region between residues 70 and 366 of ROL.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124545.g001: Sequence alignment of Rhizopus oryzae lipase (ROL) and ROL-related lipases.The full-length primary sequences of ROL, R. niveus lipase (RNL), R. stolonifer lipase (RSL), and R. chinensis lipase (RCL) are presented. Multiple-sequence alignments were generated using the ClustalW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The underlined sequences in the propeptide of ROL (Ser20–Gly37 and Ser38–Glu57) indicate the regions that are essential for secretion and folding of mROL, respectively. The underlined sequences in the mature domain of ROL (Phe183–Asp189) indicate the lid domain. The shadowed region indicates residues that were replaced with hydrophilic amino acids (VDDDDK). In the original host, R. oryzae, the propeptide is also cleaved between the Ala97 and Ser98 residues [21]; however in P. pastoris and S. cerevisiae, the secondary cleavage has not been observed [14, 22]. Therefore, in this study, we defined the propeptide domain as the region between residues 1 and 69 and the mature domain as the region between residues 70 and 366 of ROL.
Mentions: Our previous study revealed that residues Ser38–Glu57 of proROL were essential for the folding of mROL [6]. This region is highly conserved among 4 homologous lipases (Fig 1), and contains three charged amino acids (Asp41, Glu45, and Lys51) and many hydrophilic residues, indicating that proROL interacts with mROL via electrostatic and hydrophilic interactions. In particular, residues Gln50–Trp55 are highly hydrophilic. We replaced these residues with a more hydrophilic and charged amino acid sequence (VDDDDK) to alter the chaperoning function of this region. The mutated propeptide was named proROL-mut1 and the mature ROL folded by proROL-mut1 was named mROLimp (imprinted ROL) to distinguish it from mROLWT (mROL folded by wild type proROL). The two distinct mROLWT and mROLimp with the same primary sequence were produced and purified from P. pastoris (S1 Fig) using a two-step purification protocol (see Materials and Method). No apparent decrease in molecular weight was observed after endoglycosidase H (EndoH) treatment, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that mROLWT and mROLimp were not N-glycosylated. To confirm that proROL and proROL-mut1 were cleaved at the same site and that the primary sequences of mROLWT and mROLimp were identical, the protein bands were excised and analyzed by N-terminal sequencing. The analysis confirmed that both sequences of mROLWT and mROLimp started with DDNLVGGMTLD, indicating that the propeptides were cleaved at the same site and that the primary sequences of mROLWT and mROLimp were the same.

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.