Analysis of the Substrate-Recognition Mode of Aromatic Amino Acid Aminotransferase by Combined Use of Quasisubstrates and Site-Directed Mutagenesis:  Systematic Hydroxy-Group Addition/Deletion Studies to Probe the Enzyme−Substrate Interactions†

Biochemistry ◽  
1996 ◽  
Vol 35 (21) ◽  
pp. 6754-6761 ◽  
Author(s):  
Hideyuki Hayashi ◽  
Katsura Inoue ◽  
Hiroyuki Mizuguchi ◽  
Hiroyuki Kagamiyama
Keyword(s):  
Amino Acid ◽  
Hydroxy Group ◽  
Aromatic Amino Acid ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Substrate Interactions ◽  
Enzyme Substrate ◽  
Combined Use ◽  
Aromatic Amino Acid Aminotransferase

Site-directed mutagenesis of the aromatic amino acid aminotransferase of Escherichia coli

1989 ◽  
Vol 17 (1) ◽  
pp. 195-195
Author(s):  
MARTIN J. GARTLAND ◽  
MICHAEL G. HUNTER ◽  
IAN G. FOTHERINGHAM ◽  
GEOFFREY C. ROWLAND ◽  
ROBERT E. GLASS
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Aromatic Amino Acid Aminotransferase

scholarly journals A Novel FexA Variant from a Canine Staphylococcus pseudintermedius Isolate That Does Not Confer Florfenicol Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5763-5766 ◽  
Author(s):  
Elena Gómez-Sanz ◽  
Kristina Kadlec ◽  
Andrea T. Feßler ◽  
Myriam Zarazaga ◽  
Carmen Torres ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Base Pairs ◽  
Chloramphenicol Resistance ◽  
Content Type ◽  
Resistance Phenotype ◽  
First Time

ABSTRACTTransposon Tn558integrated in the chromosomalradCgene was detected for the first time inStaphylococus pseudintermedius. It carried a novelfexAvariant (fexAv) that confers only chloramphenicol resistance. The exporter FexAv exhibited two amino acid substitutions, Gly33Ala and Ala37Val, both of which seem to be important for substrate recognition. Site-directed mutagenesis that reverted the mutated base pairs to those present in the originalfexAgene restored the chloramphenicol-plus-florfenicol resistance phenotype.

scholarly journals Site-directed Mutagenesis and Kinetic Studies of the West Nile Virus NS3 Protease Identify Key Enzyme-Substrate Interactions

2004 ◽  
Vol 280 (4) ◽  
pp. 2896-2903 ◽  
Author(s):  
Keith J. Chappell ◽  
Tessa A. Nall ◽  
Martin J. Stoermer ◽  
Ning-Xia Fang ◽  
Joel D. A. Tyndall ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
The West ◽  
West Nile ◽  
Substrate Interactions ◽  
Enzyme Substrate ◽  
Key Enzyme ◽  
Ns3 Protease

Creating lipoxygenases with new positional specificities by site-directed mutagenesis

2000 ◽  
Vol 28 (6) ◽  
pp. 825-826 ◽  
Author(s):  
E. Hornung ◽  
S. Rosahl ◽  
H. Kühn ◽  
I. Feussner
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
Positional Specificity ◽  
The Past ◽  
Enzyme Substrate ◽  
Plant Lipoxygenases

In order to analyse the amino acid determinants which alter the positional specificity of plant lipoxygenases (LOXs), multiple LOX sequence alignments and structural modelling of the enzyme-substrate interactions were carried out. These alignments suggested three amino acid residues as the primary determinants of positional specificity. Here we show the generation of two plant LOXs with new positional specificities, a Δ-linoleneate 6-LOX and an arachidonate 11-LOX, by altering only one of these determinants within the active site of two plant LOXs. In the past, site-directed-mutagenesis studies have mainly been carried out with mammalian lipoxygenases (LOXs) [1]. In these experiments two regions have been identified in the primary structure containing sequence determinants for positional specificity. Amino acids aligning with the Sloane determinants [2] are highly conserved among plant LOXs. In contrast, there is amino acid hetero-geneity among plant LOXs at the position that aligns with P353 of the rabbit reticulocyte 15-LOX (Borngräber determinants) [3].

Identification of amino acid residues involved in substrate recognition of l-xylulose reductase by site-directed mutagenesis

2003 ◽  
Vol 143-144 ◽  
pp. 543-550 ◽  
Author(s):  
Shuhei Ishikura ◽  
Tomoya Isaji ◽  
Noriyuki Usami ◽  
Junichi Nakagawa ◽  
Ossama El-Kabbani ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues

scholarly journals Asymmetry in catalysis: ‘unidirectional’ amino acid racemases

2021 ◽  
Vol 43 (1) ◽  
pp. 28-34
Author(s):  
Stephen L. Bearne
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Hydroxyl Group ◽  
Directed Mutagenesis ◽  
Biotechnological Applications ◽  
Enzyme Substrate ◽  
Brønsted Base ◽  
Catalytic Dyad

d-Amino acids play widespread structural, functional and regulatory roles in organisms. These d-amino acids often arise through the stereoinversion of the more plentiful l-amino acids catalysed by amino acid racemases and epimerases. Such enzymes are of interest since many are recognized targets for the development of drugs or may be employed industrially in biotransformation reactions. Despite their enzyme–substrate complexes being diastereomers, some racemases and epimerases exhibit a kinetic pseudo-symmetry, binding their enantiomeric or epimeric substrate pairs with roughly equal affinities and catalyzing their stereoinversion with similar turnover numbers. In other cases, this kinetic pseudo-symmetry is absent or may be ‘broken’ by substitution of a catalytic Cys by Ser at the active site of cofactor-independent racemases and epimerases, or by altering the Brønsted base of the catalytic dyad that facilitates deprotonation of the Cys residue. Moreover, a natural Thr-containing l-Asp/Glu racemase was discovered that catalyses ‘unidirectional’ substrate turnover, unlike the typical bidirectional racemases and epimerases. These observations suggest that bidirectional Cys–Cys racemases may be re-engineered into ‘unidirectional’ racemases through substitution of the thiol by a hydroxyl group. Catalysis by such ‘unidirectional’ racemase precursors could then be optimized further by site-directed mutagenesis and directed evolution to furnish useful enzymes for biotechnological applications.

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