Site-Directed Mutagenesis Reveals the Thermodynamic Requirements for Single-Stranded DNA Recognition by the Telomere-Binding Protein Cdc13†

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective markerin E. coli. Vector pIAFS20 also contains the phage fl intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screeningof the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis. Key words: shuttle vector, single-stranded DNA, site-directed mutagenesis, Streptomyces spp., Escherichia coli.

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Catalytic Mechanism of theStreptomycesK15dd-Transpeptidase/Penicillin-Binding Protein Probed by Site-Directed Mutagenesis and Structural Analysis†,‡

Biochemistry ◽

10.1021/bi027256x ◽

2003 ◽

Vol 42 (10) ◽

pp. 2895-2906 ◽

Cited By ~ 12

Author(s):

Noureddine Rhazi ◽

Paulette Charlier ◽

Dominique Dehareng ◽

Danièle Engher ◽

Marcel Vermeire ◽

...

Keyword(s):

Structural Analysis ◽

Catalytic Mechanism ◽

Binding Protein ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Penicillin Binding Protein

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Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

Biochemical Journal ◽

10.1042/bj3030357 ◽

1994 ◽

Vol 303 (2) ◽

pp. 357-362 ◽

Cited By ~ 29

Author(s):

M P G van der Linden ◽

L de Haan ◽

O Dideberg ◽

W Keck

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Binding Capacity ◽

Binding Protein ◽

Complete Loss ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Penicillin Binding Protein ◽

Wild Type ◽

Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

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Turn scanning by site-directed mutagenesis: Application to the protein folding problem using the intestinal fatty acid binding protein

Protein Science ◽

10.1002/pro.5560070818 ◽

1998 ◽

Vol 7 (8) ◽

pp. 1821-1828 ◽

Cited By ~ 29

Author(s):

Keehyuk Kim ◽

Carl Frieden

Keyword(s):

Protein Folding ◽

Fatty Acid ◽

Fatty Acid Binding Protein ◽

Binding Protein ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Fatty Acid Binding ◽

Acid Binding Protein

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Transferrin-Binding Protein B of Neisseria meningitidis: Sequence-Based Identification of the Transferrin-Binding Site Confirmed by Site-Directed Mutagenesis

Journal of Bacteriology ◽

10.1128/jb.186.3.850-857.2004 ◽

2004 ◽

Vol 186 (3) ◽

pp. 850-857 ◽

Cited By ~ 8

Author(s):

Geneviève Renauld-Mongénie ◽

Laurence Lins ◽

Tino Krell ◽

Laure Laffly ◽

Michèle Mignon ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Binding Protein ◽

Prediction Method ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Titration Calorimetry ◽

Directed Mutagenesis ◽

Binding Domains ◽

Homologous Strain ◽

Protein B

ABSTRACT A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

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