Soluble Overexpression inEscherichia coli,and Purification and Characterization of Wild-Type Recombinant Tobacco Acetolactate Synthase

Chang, Soo-Ik; Kang, Moon-Kyeong; Choi, Jung‐Do; Namgoong, Sung Keon

doi:10.1006/bbrc.1997.6678

Cited by 37 publications

(13 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hence, MBP probably served as a suitable molecular chaperone that supported the proper folding of fp1 for soluble expression. In contrast, GST has failed to produce soluble fp1 although it has been reported to enable soluble expression of proteins that tend to form inclusion aggregates (Chang et al 1997). This is congruent with the studies by Kapust and Waugh (1999), which showed that MBP to be a better solubilizing agent than GST and TRX.…”

Section: Discussionsupporting

confidence: 59%

Cysteine protease falcipain 1 in Plasmodium falciparum is biochemically distinct from its isozymes

Goh

Sim

2005

Parasitol Res

View full text Add to dashboard Cite

Falcipains form a class of papain-like cysteine proteases found in Plasmodium falciparum. This group of proteases has been suggested to be promising targets for anti-malarial chemotherapy. Despite being the first falcipain to be identified, the physiological role(s) of falcipain 1 (fp1) remains a mystery. Its suggested functions include haemoglobin degradation, erythrocytic invasion and oocyst production. In this study, the procurement of the gene coding for fp1 and its soluble expression in a heterologous host, Escherichia coli, have enabled further enzyme characterization. The recombinant fp1 protease was found to be unlike falcipain 2 (fp2A) in being more active at neutral pH than at acidic pH against the Z-LR-AMC fluorogenic substrate, suggesting a probable localization in the cytosol and not in the food vacuole. Interestingly, a common cysteine specific inhibitor, E64, did not inhibit fp1 activity, indicating dissimilar biochemical characteristics of fp1 from the other falcipains. This may be explained by computational analysis of the primary structures of the falcipain isozymes, as well as that of papain. The analysis revealed that Tyr61 (papain numbering), which is correspondingly absent in fp1, might be an important residue involved in E64 substrate binding.

show abstract

Section: Discussionsupporting

confidence: 59%

Cysteine protease falcipain 1 in Plasmodium falciparum is biochemically distinct from its isozymes

Goh

Sim

2005

Parasitol Res

View full text Add to dashboard Cite

show abstract

“…Expression and purification of the recombinant AHAS was performed with modification as described by Chang et al [29]. Briefly, Escherichia coli DH5α cells containing the expression vector pGEX‐ALS were grown at 37°C in Luria–Bertani medium containing 50 μg/ml ampicillin to an OD 600 of 0.7–0.8.…”

Section: Methodsmentioning

confidence: 99%

The active site and mechanism of action of recombinant acetohydroxy acid synthase from tobacco

et al. 2003

Self Cite

View full text Add to dashboard Cite

Acetohydroxy acid synthase (AHAS) is one of several enzymes that require thiamine diphosphate and a divalent cation as essential cofactors. Recently, the three-dimensional structure of the enzyme from yeast has been determined [Pang et al., J. Mol. Biol. 317 (2002) 249-262]. While this structure sheds light on the binding of the cofactors and the reaction mechanism, the interactions between the substrates and the enzyme remain unclear. We have studied the pH dependence of kinetic parameters in order to obtain information about the chemical mechanism in the active site. Data are consistent with a mechanism in which substrate selectively catalyzed to the enzyme with an unprotonated base having a pK of 6.48, and a protonated group having a pK of 8.25 for catalysis. The temperature dependence of kinetic parameters was pH-dependent, and the enthalpies of ionization, DeltaH(ion), calculated from the slope of pK(1) and pK(2) are both pH-independent. The solvent perturbation of kinetic parameters was pH-dependent, and the pK(1) from the acidic side and the pK(2) from the basic side were shifted down 0.4 pH units and shifted up 0.6 units as water was replaced by 15% ethanol, respectively. The data are discussed in terms of the acid-base chemical mechanism.

show abstract

“…To determine the effects of these deletions on enzyme activity, we measured the enzyme activity under the standard saturating concentration of cofactors (1 mM ThDP, 20 µM FAD and 10 mM Mg 2+ ) and substrate (100 mM pyruvate) as reported previously [24,25] (Table 1). All of the deletions greatly reduced the activity to that of a few percent of wild-type enzyme, and deletion of the entire C-terminus ( 567), including both mobile loop and C-terminal lid, did not show any detectable activity.…”

Section: Construction and Expression Of C-terminal Truncated Tobacco mentioning

confidence: 99%

Effects of deletions at the C-terminus of tobacco acetohydroxyacid synthase on the enzyme activity and cofactor binding

Kim

Beak

Kim

et al. 2004

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

AHAS (acetohydroxyacid synthase) catalyses the first committed step in the biosynthesis of branched-chain amino acids, such as valine, leucine and isoleucine. Owing to the unique presence of these biosynthetic pathways in plants and micro-organisms, AHAS has been widely investigated as an attractive target of several classes of herbicides. Recently, the crystal structure of the catalytic subunit of yeast AHAS has been resolved at 2.8 A (1 A=0.1 nm), showing that the active site is located at the dimer interface and is near the herbicide-binding site. In this structure, the existence of two disordered regions, a 'mobile loop' and a C-terminal 'lid', is worth notice. Although these regions contain the residues that are known to be important in substrate specificity and in herbicide resistance, they are poorly folded into any distinct secondary structure and are not within contact distance of the cofactors. In the present study, we have tried to demonstrate the role of these regions of tobacco AHAS by constructing variants with serial deletions, based on the structure of yeast AHAS. In contrast with the wild-type AHAS, the truncated mutant which removes the C-terminal lid, Delta630, and the internal deletion mutant without the mobile loop, Delta567-582, impaired the binding affinity for ThDP (thiamine diphosphate), and showed different elution profiles representing a monomeric form in gel-filtration chromatography. Our results suggest that these regions are involved in the binding/stabilization of the active dimer and ThDP binding.

show abstract

Soluble Overexpression inEscherichia coli,and Purification and Characterization of Wild-Type Recombinant Tobacco Acetolactate Synthase

Cited by 37 publications

References 30 publications

Cysteine protease falcipain 1 in Plasmodium falciparum is biochemically distinct from its isozymes

Cysteine protease falcipain 1 in Plasmodium falciparum is biochemically distinct from its isozymes

The active site and mechanism of action of recombinant acetohydroxy acid synthase from tobacco

Effects of deletions at the C-terminus of tobacco acetohydroxyacid synthase on the enzyme activity and cofactor binding

Contact Info

Product

Resources

About