Using X-ray crystallography of the Asp55Asn mutant of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus to support the mechanistic role of Asp55 as the general base

Nina Goodey, Arthur F. Monzingo, Christopher L. Franklin, Jon D. Robertus, Stephen F. Martin

Research output: Contribution to journalArticleResearchpeer-review

10 Citations (Scopus)

Abstract

Because mutations of the ionizable Asp at position 55 of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLCBc) to a non-ionizable Asn generate a mutant enzyme (D55N) with 104-fold lower catalytic activity than the wild-type enzyme, we tentatively identified Asp55 as the general base for the enzymatic reaction. To eliminate the alternate possibility that Asp55 is a structurally important amino acid, the X-ray structures of unbound D55N and complexes of D55N with two non-hydrolyzable substrate analogues have been solved and refined to 2.0, 2.0, and 2.3Å, respectively. The structures of unbound wild-type PLCBc and a wild-type PLCBc-complex with a non-hydrolyzable substrate analogue do not change significantly as a result of replacing Asp55 with Asn. These observations demonstrate that Asp55 is not critical for the structural integrity of the enzyme and support the hypothesis that Asp55 is the general base in the PLCBc-catalyzed hydrolysis of phospholipids.

Original languageEnglish
Pages (from-to)81-86
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume417
Issue number1
DOIs
StatePublished - 1 Sep 2003

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Bacillus cereus
X ray crystallography
X Ray Crystallography
Type C Phospholipases
Phosphatidylcholines
Enzymes
Structural integrity
Substrates
Hydrolysis
Catalyst activity
Phospholipids
X-Rays
Amino Acids
X rays
Mutation

Keywords

  • Phospholipase C from Bacillus cereus
  • Site-directed mutagenesis

Cite this

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title = "Using X-ray crystallography of the Asp55Asn mutant of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus to support the mechanistic role of Asp55 as the general base",
abstract = "Because mutations of the ionizable Asp at position 55 of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLCBc) to a non-ionizable Asn generate a mutant enzyme (D55N) with 104-fold lower catalytic activity than the wild-type enzyme, we tentatively identified Asp55 as the general base for the enzymatic reaction. To eliminate the alternate possibility that Asp55 is a structurally important amino acid, the X-ray structures of unbound D55N and complexes of D55N with two non-hydrolyzable substrate analogues have been solved and refined to 2.0, 2.0, and 2.3{\AA}, respectively. The structures of unbound wild-type PLCBc and a wild-type PLCBc-complex with a non-hydrolyzable substrate analogue do not change significantly as a result of replacing Asp55 with Asn. These observations demonstrate that Asp55 is not critical for the structural integrity of the enzyme and support the hypothesis that Asp55 is the general base in the PLCBc-catalyzed hydrolysis of phospholipids.",
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journal = "Archives of Biochemistry and Biophysics",
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Using X-ray crystallography of the Asp55Asn mutant of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus to support the mechanistic role of Asp55 as the general base. / Goodey, Nina; Monzingo, Arthur F.; Franklin, Christopher L.; Robertus, Jon D.; Martin, Stephen F.

In: Archives of Biochemistry and Biophysics, Vol. 417, No. 1, 01.09.2003, p. 81-86.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Using X-ray crystallography of the Asp55Asn mutant of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus to support the mechanistic role of Asp55 as the general base

AU - Goodey, Nina

AU - Monzingo, Arthur F.

AU - Franklin, Christopher L.

AU - Robertus, Jon D.

AU - Martin, Stephen F.

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N2 - Because mutations of the ionizable Asp at position 55 of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLCBc) to a non-ionizable Asn generate a mutant enzyme (D55N) with 104-fold lower catalytic activity than the wild-type enzyme, we tentatively identified Asp55 as the general base for the enzymatic reaction. To eliminate the alternate possibility that Asp55 is a structurally important amino acid, the X-ray structures of unbound D55N and complexes of D55N with two non-hydrolyzable substrate analogues have been solved and refined to 2.0, 2.0, and 2.3Å, respectively. The structures of unbound wild-type PLCBc and a wild-type PLCBc-complex with a non-hydrolyzable substrate analogue do not change significantly as a result of replacing Asp55 with Asn. These observations demonstrate that Asp55 is not critical for the structural integrity of the enzyme and support the hypothesis that Asp55 is the general base in the PLCBc-catalyzed hydrolysis of phospholipids.

AB - Because mutations of the ionizable Asp at position 55 of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLCBc) to a non-ionizable Asn generate a mutant enzyme (D55N) with 104-fold lower catalytic activity than the wild-type enzyme, we tentatively identified Asp55 as the general base for the enzymatic reaction. To eliminate the alternate possibility that Asp55 is a structurally important amino acid, the X-ray structures of unbound D55N and complexes of D55N with two non-hydrolyzable substrate analogues have been solved and refined to 2.0, 2.0, and 2.3Å, respectively. The structures of unbound wild-type PLCBc and a wild-type PLCBc-complex with a non-hydrolyzable substrate analogue do not change significantly as a result of replacing Asp55 with Asn. These observations demonstrate that Asp55 is not critical for the structural integrity of the enzyme and support the hypothesis that Asp55 is the general base in the PLCBc-catalyzed hydrolysis of phospholipids.

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