Site-directed mutagenesis enabled preparation of a functional fluorescent analog of profilin: Biochemical characterization and localization in living cells

ABSTRACT Three mutants of the extended-spectrum β-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis. The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.

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Involvement of various amino- and carboxyl-terminal residues in the active site of the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus: site-directed mutagenesis with the ptsH gene, biochemical characterization and NMR studies of the mutant proteins

Protein Engineering Design and Selection ◽

10.1093/protein/6.4.417 ◽

1993 ◽

Vol 6 (4) ◽

pp. 417-423 ◽

Cited By ~ 12

Author(s):

R. Kruse ◽

W. Hengstenberg ◽

W. Beneicke ◽

H.R. Kalbitzer

Keyword(s):

Active Site ◽

Biochemical Characterization ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Phosphotransferase System ◽

Nmr Studies ◽

Mutant Proteins ◽

Staphylococcus Carnosus ◽

Carboxyl Terminal

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Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin

Protein Engineering Design and Selection ◽

10.1093/protein/9.10.885 ◽

1996 ◽

Vol 9 (10) ◽

pp. 885-893 ◽

Cited By ~ 6

Author(s):

P.G. Nugent ◽

A. Albert ◽

P. Orprayoon ◽

J. Wilsher ◽

J.E. Pitts ◽

...

Keyword(s):

Protein Engineering ◽

Biochemical Characterization ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Aspartic Proteinases ◽

X Ray

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Identification of Residues Important for the Activity ofHaloferax volcaniiAglD, a Component of the Archaeal N-Glycosylation Pathway

Archaea ◽

10.1155/2010/315108 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Lina Kaminski ◽

Jerry Eichler

Keyword(s):

Biochemical Characterization ◽

Haloferax Volcanii ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Natural Substrate ◽

Identify Amino Acid ◽

Glycosylation Pathway

InHaloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in anHfx. volcanii aglDdeletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

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