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 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 Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

2002 ◽  
Vol 46 (9) ◽  
pp. 3035-3038 ◽  
Author(s):  
Barry G. Hall
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Dna Shuffling ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Single Mutation ◽  
Wild Type ◽  
In Vitro Evolution ◽  
Evolutionary Pathway

ABSTRACT In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

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 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.

Sign in / Sign up

Export Citation Format

Share Document