scholarly journals Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate

2010 ◽  
Vol 76 (17) ◽  
pp. 6001-6005 ◽  
Author(s):  
Yong-Sheng Tian ◽  
Ai-Sheng Xiong ◽  
Jing Xu ◽  
Wei Zhao ◽  
Feng Gao ◽  
...  
Keyword(s):  
Genomic Library ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Construction Process ◽  
Ochrobactrum Anthropi ◽  
Gene Encoding ◽  
Phosphate Synthase

ABSTRACT Applying the genomic library construction process and colony screening, a novel aro A gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.

scholarly journals Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis

2002 ◽  
Vol 365 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Antonella De LUCA ◽  
Bartolo FAVALORO ◽  
Stefania ANGELUCCI ◽  
Paolo SACCHETTA ◽  
Carmine Di ILIO
Keyword(s):  
Xenopus Laevis ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Structural Instability ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

scholarly journals Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis

1997 ◽  
Vol 323 (1) ◽  
pp. 61-64 ◽  
Author(s):  
Kazuya MATSUURA ◽  
Yoshihiro DEYASHIKI ◽  
Kumiko SATO ◽  
Naoko ISHIDA ◽  
Gunpei MIWA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Human Liver ◽  
Hydroxysteroid Dehydrogenase ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Dihydrodiol Dehydrogenase ◽  
Inactive Protein

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20α-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3α-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors.

scholarly journals Function of the 90-loop (Thr90–Glu100) region of staphylokinase in plasminogen activation probed through site-directed mutagenesis and loop deletion

2002 ◽  
Vol 365 (2) ◽  
pp. 379-389 ◽  
Author(s):  
Govindan RAJAMOHAN ◽  
Monika DAHIYA ◽  
Shekhar C. MANDE ◽  
Kanak L. DIKSHIT
Keyword(s):  
Active Site ◽  
Complex Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Kringle Domains ◽  
Lysine Residues ◽  
Modelling Studies ◽  
Activator Complex

Staphylokinsae (SAK) forms a bimolecular complex with human plasmin(ogen) and changes its substrate specificity by exposing new exosites that enhances accession of substrate plasminogen (PG) to the plasmin (Pm) active site. Protein modelling studies indicated the crucial role of a loop in SAK (SAK 90-loop; Thr90—Glu100) for the docking of the substrate PG to the SAK—Pm complex. Function of SAK 90-loop was studied by site-directed mutagenesis and loop deletion. Deletion of nine amino acid residues (Tyr92—Glu100) from the SAK 90-loop, resulted in ≈60% reduction in the PG activation, but it retained the ability to generate an active site within the complex of loop mutant of SAK (SAKΔ90) and Pm. The preformed activator complex of SAKΔ90 with Pm, however, displayed a 50–60% reduction in substrate PG activation that remained unaffected in the presence of kringle domains (K1+K2+K3+K4) of PG, whereas PG activation by SAK—Pm complex displayed ∼50% reduction in the presence of kringles, suggesting the involvement of the kringle domains in modulating the PG activation by native SAK but not by SAKΔ90. Lysine residues (Lys94, Lys96, Lys97 and Lys98) of the SAK 90-loop were individually mutated into alanine and, among these four SAK loop mutants, SAKK97A and SAKK98A exhibited specific activities about one-third and one-quarter respectively of the native SAK. The kinetic parameters of PG activation of their 1:1 complex with Pm indicated that the Km values of PG towards the activator complex of these two SAK mutants were 4–6-fold higher, suggesting the decreased accessibility of the substrate PG to the activator complex formed by these SAK mutants. These results demonstrated the involvement of the Lys97 and Lys98 residues of the SAK 90-loop in assisting the interaction with substrate PG. These interactions of SAK—Pm activator complex via the SAK 90-loop may provide additional anchorage site(s) to the substrate PG that, in turn, may promote the overall process of SAK-mediated PG activation.

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