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Mutagenesis Studies of Substrate Recognition and Catalysis in the Sortase A Transpeptidase from Staphylococcus aureus*

Bentley, Matthew L.; Lamb, Erin C.; McCafferty, Dewey G.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 23/05/2008 Português
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36.64%
The Staphylococcus aureus transpeptidase sortase A (SrtA) is responsible for anchoring a range of virulence- and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the β6/β7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca2+-binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in kcat/Km, indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169...

Force-Clamp Spectroscopy Detects Residue Co-evolution in Enzyme Catalysis*S⃞

Perez-Jimenez, Raul; Wiita, Arun P.; Rodriguez-Larrea, David; Kosuri, Pallav; Gavira, Jose A.; Sanchez-Ruiz, Jose M.; Fernandez, Julio M.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 03/10/2008 Português
Relevância na Pesquisa
36.72%
Understanding how the catalytic mechanisms of enzymes are optimized through evolution remains a major challenge in molecular biology. The concept of co-evolution implicates that compensatory mutations occur to preserve the structure and function of proteins. We have combined statistical analysis of protein sequences with the sensitivity of single molecule force-clamp spectroscopy to probe how catalysis is affected by structurally distant correlated mutations in Escherichia coli thioredoxin. Our findings show that evolutionary anti-correlated mutations have an inhibitory effect on enzyme catalysis, whereas positively correlated mutations rescue the catalytic activity. We interpret these results in terms of an evolutionary tuning of both the enzyme-substrate binding process and the chemistry of the active site. Our results constitute a direct observation of distant residue co-evolution in enzyme catalysis.

Escherichia coli DNA Adenine Methyltransferase: THE STRUCTURAL BASIS OF PROCESSIVE CATALYSIS AND INDIRECT READ-OUT

Coffin, Stephanie R.; Reich, Norbert O.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.72%
We have investigated the structural basis of processive GATC methylation by the Escherichia coli DNA adenine methyltransferase, which is critical in chromosome replication and mismatch repair. We determined the contribution of the orthologically conserved phosphate interactions involving residues Arg95, Asn126, Asn132, Arg116, and Lys139, which directly contact the DNA outside the cognate recognition site (GATC) to processive catalysis, and that of residue Arg137, which is not conserved and contacts the DNA backbone within the GATC sequence. Alanine substitutions at the conserved positions have large impacts on processivity yet do not impact kcat/KmDNA or DNA affinity (KDDNA). However, these mutants cause large preferences for GATC sites varying in flanking sequences when considering the pre-steady state efficiency constant kchem/KDDNA. These changes occur mainly at the level of the methylation rate constant, which results in the observed decreases in processive catalysis. Thus, processivity and catalytic efficiency (kcat/KmDNA) are uncoupled in these mutants. These results reveal that the binding energy involved in DNA recognition contributes to the assembly of the active site rather than tight binding. Furthermore, the conserved residues (Arg95...