Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase

Biochemistry ◽  
1989 ◽  
Vol 28 (2) ◽  
pp. 505-509 ◽  
Author(s):  
M. Merola ◽  
A. Martinez del Pozo ◽  
H. Ueno ◽  
P. Recsei ◽  
A. Di Donato ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Serine Residue

scholarly journals Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

2014 ◽  
Vol 70 (4) ◽  
pp. 1166-1172 ◽  
Author(s):  
Michael E. Webb ◽  
Briony A. Yorke ◽  
Tom Kershaw ◽  
Sarah Lovelock ◽  
Carina M. C. Lobley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intramolecular Rearrangement ◽  
Directed Mutagenesis ◽  
Vitamin B ◽  
Biosynthesis Pathway ◽  
Serine Residue ◽  
Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis

Biochimie ◽  
2007 ◽  
Vol 89 (12) ◽  
pp. 1498-1508 ◽  
Author(s):  
Muriel Crouvoisier ◽  
Geneviève Auger ◽  
Didier Blanot ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Site Directed Mutagenesis of Amino Acid Residues at the Active Site of Mouse Aldehyde Oxidase AOX1

PLoS ONE ◽  
2009 ◽  
Vol 4 (4) ◽  
pp. e5348 ◽  
Author(s):  
Silvia Schumann ◽  
Mineko Terao ◽  
Enrico Garattini ◽  
Miguel Saggu ◽  
Friedhelm Lendzian ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Aldehyde Oxidase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues

scholarly journals Structural Modeling and Site-Directed Mutagenesis of the Actinorhodin β-Ketoacyl-Acyl Carrier Protein Synthase

2000 ◽  
Vol 182 (9) ◽  
pp. 2619-2623 ◽  
Author(s):  
Min He ◽  
Mustafa Varoglu ◽  
David H. Sherman
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Three Dimensional Model ◽  
Catalytic Active Site ◽  
Ketoacyl Synthase

ABSTRACT A three-dimensional model of the Streptomyces coelicolor actinorhodin β-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the relatedEscherichia coli fatty acid synthase condensing enzyme β-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production. 3H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.

Investigating the active site of human trimethyllysine hydroxylase

2019 ◽  
Vol 476 (7) ◽  
pp. 1109-1119 ◽  
Author(s):  
Yali Wang ◽  
Y. Vijayendar Reddy ◽  
Abbas H. K. Al Temimi ◽  
Hanka Venselaar ◽  
Frank H. T. Nelissen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Biosynthesis Pathway ◽  
Recognition Sites ◽  
Aromatic Cage ◽  
Basic Insight ◽  
Insight Into

Abstract The biologically important carnitine biosynthesis pathway in humans proceeds via four enzymatic steps. The first step in carnitine biosynthesis is catalyzed by trimethyllysine hydroxylase (TMLH), a non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase, which catalyzes the stereospecific hydroxylation of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. Here, we report biocatalytic studies on human TMLH and its 19 variants introduced through site-directed mutagenesis. Amino acid substitutions at the sites involved in binding of the Fe(II) cofactor, 2OG cosubstrate and (2S)-Nε-trimethyllysine substrate provide a basic insight into the binding requirements that determine an efficient TMLH-catalyzed conversion of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. This work demonstrates the importance of the recognition sites that contribute to the enzymatic activity of TMLH: the Fe(II)-binding H242–D244–H389 residues, R391–R398 involved in 2OG binding and several residues (D231, N334 and the aromatic cage comprised of W221, Y217 and Y234) associated with binding of (2S)-Nε-trimethyllysine.

scholarly journals Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis

2002 ◽  
Vol 367 (3) ◽  
pp. 781-789 ◽  
Author(s):  
Nobutaka FUNA ◽  
Yasuo OHNISHI ◽  
Yutaka EBIZUKA ◽  
Sueharu HORINOUCHI
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Polyketide Synthase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Type Iii Polyketide Synthase ◽  
Melanin Biosynthesis ◽  
Type Iii ◽  
Malonyl Coa

RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys138, His270 and Asn303 of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr224 is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr224 by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala305 mutants and identification of their products showed that the substitution of Ala305 by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr224 (important for starter substrate selection) and Ala305 (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.

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