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Exploring structure-function of the ...

Wymer, Nathan John.

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Exploring structure-function of the KDPG aldolase with site-directed mutagenesis and directed evolution.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Exploring structure-function of the KDPG aldolase with site-directed mutagenesis and directed evolution./
Author:	Wymer, Nathan John.
Description:	128 p.
Notes:	Source: Dissertation Abstracts International, Volume: 63-09, Section: B, page: 4183.
Contained By:	Dissertation Abstracts International63-09B.
Subject:	Biology, Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3063212
ISBN:	0493819681

Exploring structure-function of the KDPG aldolase with site-directed mutagenesis and directed evolution.
Wymer, Nathan John.

Exploring structure-function of the KDPG aldolase with site-directed mutagenesis and directed evolution. - 128 p.

Source: Dissertation Abstracts International, Volume: 63-09, Section: B, page: 4183.

Thesis (Ph.D.)--Duke University, 2002.

This thesis covers the efforts to expand the synthetic potential of the <italic>Eschericia coli</italic> 2-keto-3-deoxy-6-phosphogluconate aldolase (E.C. 4.1.2.14). The effort focused on the understanding of amino acid structure relationships as they relate to specific activity and in modifying the substrate specificity of the aldolase to include hydrophobic electrophiles, i.e. benzaldehyde. The inclusion of benzaldehyde as a substrate would facilitate the enantioselective synthesis of thiamphenicol and fluorphenicol, two broad spectrum antibiotics. This change in substrate specificity was accomplished with a combination of directed evolution and site-directed mutagenesis.

ISBN: 0493819681Subjects--Topical Terms:

1017734
Biology, Microbiology.

Exploring structure-function of the KDPG aldolase with site-directed mutagenesis and directed evolution.
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020 $a 0493819681
035 $a (UnM)AAI3063212
035 $a AAI3063212
040 $a UnM $c UnM
100 1 $a Wymer, Nathan John. $3 1251354
245 1 0 $a Exploring structure-function of the KDPG aldolase with site-directed mutagenesis and directed evolution.
300 $a 128 p.
500 $a Source: Dissertation Abstracts International, Volume: 63-09, Section: B, page: 4183.
500 $a Supervisor: Eric J. Toone.
502 $a Thesis (Ph.D.)--Duke University, 2002.
520 $a This thesis covers the efforts to expand the synthetic potential of the <italic>Eschericia coli</italic> 2-keto-3-deoxy-6-phosphogluconate aldolase (E.C. 4.1.2.14). The effort focused on the understanding of amino acid structure relationships as they relate to specific activity and in modifying the substrate specificity of the aldolase to include hydrophobic electrophiles, i.e. benzaldehyde. The inclusion of benzaldehyde as a substrate would facilitate the enantioselective synthesis of thiamphenicol and fluorphenicol, two broad spectrum antibiotics. This change in substrate specificity was accomplished with a combination of directed evolution and site-directed mutagenesis.
520 $a Residues 131 to 168 were explored with directed evolution and three mutants were identified: P160P, T161K, N168S. P160P is a silent mutation that provides better codon usage. In order to understand the significance of the two other mutants, the crystal structure of the <italic>E. coli</italic> aldolase was solved. The N168S mutation is located outside the active site and was not further studied. The T161K mutation creates a new lysine within the active site and was theorized to form a new catalytic lysine. To test this theory, a K133Q mutant was prepare to remove the original catalytic lysine. The K133Q mutation had no catalytic activity. Preparation of the T161K/K133Q mutant recovered the aldolase activity. Kinetic studies and pyruvate labeling experiments showed that the T161K mutation was acting as a new catalytic lysine.
520 $a The repositioning of the catalytic lysine with the T161K/K133Q mutant aldolase comes at the expense of catalyic activity. The T161K/K133Q mutant showed a drop in kcat of 1000-fold for both KDPG and 2-pyridine carboxaldehyde, compared to wild-type aldolase. However, the T161K/K133Q mutant did show a shift in substrate specificity to include benzaldehyde.
520 $a With the success of the T161K/K133Q mutant, V20K/K133Q and G182K/K133Q mutant aldolases were prepared to position the catalytic lysine around the active site. The V20K/K133Q and G182K/K133Q showed similar 1000-fold drops in kinetic rates compared to wild-type. However, these two moved lysine mutants showed broader substrate specificities than either the T161K/K133Q mutant aldolase or wild-type aldolase.
520 $a In order to probe various mechanistic postulates, several conserved residues within the active site were also mutated to monitor effects on substrate specificities and kinetic rates. R49Q, T73A, F135I, and E45N mutants were prepared, and all show broader substrate specificities compared to wild-type aldolase, with the exception of R49Q. However, all of these mutants do accept benzaldehyde as an electrophile. The R49Q and T73A mutants have the highest reaction rates with only a 10-fold drop in kcat for 2-pyridine carboxaldehyde compared to wild-type aldolase. The F135I and E45N mutants have a 100-fold and 1000-fold decrease in kcat respectively for 2-pyridine carboxaldehyde compared to wild-type aldolase. Overall, the combination of site-directed mutagenesis and directed evolution was successfully modified the substrate specificity of the KDPG aldolase.
590 $a School code: 0066.
650 4 $a Biology, Microbiology. $3 1017734
650 4 $a Chemistry, Biochemistry. $3 1017722
650 4 $a Chemistry, Organic. $3 516206
690 $a 0410
690 $a 0487
690 $a 0490
710 2 0 $a Duke University. $3 569686
773 0 $t Dissertation Abstracts International $g 63-09B.
790 $a 0066
790 1 0 $a Toone, Eric J., $e advisor
791 $a Ph.D.
792 $a 2002
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3063212