Structure analysis of endosialidase NF at 0.98 Å resolution

2010 ◽  
Vol 66 (2) ◽  
pp. 176-180 ◽  
Author(s):  
Eike C. Schulz ◽  
Piotr Neumann ◽  
Rita Gerardy-Schahn ◽  
George M. Sheldrick ◽  
Ralf Ficner
Keyword(s):  
Sialic Acid ◽  
Structure Analysis ◽  
Active Site ◽  
Polysialic Acid ◽  
Crystal Form ◽  
Atomic Resolution ◽  
Active Site Residue ◽  
Catalytic Function ◽  
Multiple Conformations ◽  
A Minor

Endosialidase NF (endoNF) is a bacteriophage-derived endosialidase that specifically degrades α-2,8-linked polysialic acid. The structure of a new crystal form of endoNF in complex with sialic acid has been refined at 0.98 Å resolution. The 210 kDa homotrimeric multi-domain enzyme displays outstanding stability and resistance to SDS. Even at atomic resolution, only a minor fraction of side chains possess alternative conformations. However, multiple conformations of an active-site residue imply that it has an important catalytic function in the cleavage mechanism of polysialic acid.

scholarly journals Two β-lactamase variants with reduced clavulanic acid inhibition display different millisecond dynamics

2021 ◽  
Author(s):  
Wouter Elings ◽  
Aleksandra Chikunova ◽  
Danny B. van Zanten ◽  
Ralphe Drenth ◽  
Misbha Ud Din Ahmad ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Clavulanic Acid ◽  
Active Site ◽  
Dynamic Properties ◽  
Nmr Relaxation ◽  
Peptide Bond ◽  
Functional Screening ◽  
Catalytic Function ◽  
Multiple Conformations ◽  
Inhibitor Resistance

The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer most inhibitor resistance, K234R, as well as variant G132N that was found previously, were characterized using X-ray crystallography and NMR relaxation experiments to probe structural and dynamic properties. The G132N mutant exists in solution in two, almost equally populated conformations that exchange with a rate of ca. 88 s−1. The conformational change affects a broad region of the enzyme. The crystal structure reveals that the Asn132 side chain forces the peptide bond between Ser104 and Ile105 in a cis-conformation. The crystal structure suggests multiple conformations for several side chains (e.g. Ser104, Ser130) and a short loop (214-216). In the K234R mutant, the active site dynamics are significantly diminished with respect to the wild type enzyme. These results show that multiple evolutionary routes are available to increase inhibitor resistance in BlaC and that active site dynamics on the millisecond time scale are not required for catalytic function.

Active-site residue, domain and module swaps in modular polyketide synthases

2003 ◽  
Vol 30 (8) ◽  
pp. 489-494 ◽  
Author(s):  
Francesca Del Vecchio ◽  
Hrvoje Petkovic ◽  
Steven G. Kendrew ◽  
Lindsey Low ◽  
Barrie Wilkinson ◽  
...  
Keyword(s):  
Active Site ◽  
Polyketide Synthases ◽  
Active Site Residue

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

In Vivo Consequences of Putative Active Site Mutations in Yeast DNA Polymerases α, ε, δ, and ζ

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 47-64 ◽  
Author(s):  
Youri I Pavlov ◽  
Polina V Shcherbakova ◽  
Thomas A Kunkel
Keyword(s):  
Active Site ◽  
Dna Polymerases ◽  
Base Substitution ◽  
Loss Of Function ◽  
Chromosomal Dna ◽  
Strong Base ◽  
Dna Mismatch ◽  
Catalytic Function ◽  
Base Substitutions

Abstract Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases α, δ, ε, and ζ and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Polδ or Polε are inviable. Strains with same substitution in Rev3, the catalytic subunit of Polζ, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Polα (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Polα, -δ, or -ε are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Polα mutant has no obvious phenotype. The Polε (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Polδ mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly. Mutation rates in the Polα, -δ, and -ε mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene.

Active site residue involvement in monoamine or diamine oxidation catalysed by pea seedling amine oxidase

FEBS Journal ◽  
2011 ◽  
Vol 278 (8) ◽  
pp. 1232-1243 ◽  
Author(s):  
Maria Luisa Di Paolo ◽  
Michele Lunelli ◽  
Monika Fuxreiter ◽  
Adelio Rigo ◽  
Istvan Simon ◽  
...  
Keyword(s):  
Active Site ◽  
Amine Oxidase ◽  
Active Site Residue ◽  
Pea Seedling

scholarly journals The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B

FEBS Letters ◽  
2000 ◽  
Vol 475 (3) ◽  
pp. 157-162 ◽  
Author(s):  
Jianxing Song ◽  
Ping Xu ◽  
Hui Xiang ◽  
Zhengding Su ◽  
Andrew C. Storer ◽  
...  
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Active Site Residue ◽  
Neutral Ph ◽  
Ph Inactivation ◽  
Human Cathepsin

scholarly journals Phospholipase A2 Engineering. Structural and Functional Roles of the Highly Conserved Active Site Residue Aspartate-49

Biochemistry ◽  
1994 ◽  
Vol 33 (49) ◽  
pp. 14714-14722 ◽  
Author(s):  
Yishan Li ◽  
Bao-Zhu Yu ◽  
Hongxin Zhu ◽  
Mahendra K. Jain ◽  
Ming-Daw Tsai
Keyword(s):  
Phospholipase A2 ◽  
Active Site ◽  
Active Site Residue ◽  
Functional Roles

Identification of Glutamic Acid 381 as a Candidate Active Site Residue ofPseudomonas aeruginosaExoenzyme S†

Biochemistry ◽  
1996 ◽  
Vol 35 (8) ◽  
pp. 2754-2758 ◽  
Author(s):  
Suyan Liu ◽  
Scott M. Kulich ◽  
Joseph T. Barbieri
Keyword(s):  
Glutamic Acid ◽  
Active Site ◽  
Active Site Residue
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