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

Kim, Joungmok; Beak, Dong Gil; Kim, Young Tae; Choi, Jung Do; Yoon, Moon‐Young

doi:10.1042/bj20040427

Cited by 16 publications

(8 citation statements)

References 41 publications

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“…In AHAS, the helixloop as well as the C terminus is disordered in the holo structure (22), but in complex with several different herbicides these regions become ordered and cover the active site (54). This finding on AHAS was followed up in a recent study where the "mobile" loop and C-terminal "lid" were deleted and shown to be important for stabilization of the active dimer and ThDP binding (55). It is probable that, during the catalysis of oxalyl-CoA decarboxylation, the mobile loop in OXC changes conformation, and the 16 C-terminal disordered residues fold up and form a lid over the opening to the active site, forming the required hydrophobic environment.…”

Section: Resultsmentioning

confidence: 76%

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

Berthold

Moussatche

Richards

et al. 2005

Journal of Biological Chemistry

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Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 Å resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three ␣/␤-type domains, commonly seen in this class of enzymes, and the thiamin diphosphatebinding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.Oxalic acid is one of nature's most highly oxidized organic compounds, and its dianion is a strong chelator of metal cations, especially Ca 2ϩ , causing oxalate to be highly toxic to many organisms (1). In humans, elevated levels of oxalate are associated with several diseases, including the formation of calcium oxalate stones in the kidney (urolithiasis), renal failure, cardiomyopathy, and cardiac conductance disorders (1-3). Relatively large amounts of oxalate are introduced into the body through the diet, although this diacid may also arise as a byproduct of normal cellular metabolism (4). Because humans, in common with other mammals, are not able to degrade oxalate, this compound must be eliminated by excretion in the urine or via the intestine (5). The recent observation that a symbiotic, gut-dwelling bacterium, Oxalobacter formigenes, may regulate oxalate homeostasis in humans, therefore, has important implications for efforts to develop new strategies for treating oxalate-related diseases (6). O. formigenes is an obligate anaerobe bacterium found in the gastrointestinal tracts of vertebrates, including humans, and is unusual in that it employs oxalate as the sole energy source for its survival (7,8). As a result, this bacterium not only degrades free oxalate entering the intestine lumen but also creates a transepithelial gradient favoring oxalate secretion and preventing absorption of oxalic acid in the lower tract of the intestine (9). A direct correlation between the number of recurrent kidney stone episodes and a lack of O. formigenes in the ...

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Section: Resultsmentioning

confidence: 76%

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

Berthold

Moussatche

Richards

et al. 2005

Journal of Biological Chemistry

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“…7), several amino acid residues including G, Q, L, I, N, S, D, K are conserved throughout. All these amino acids belong to C terminal domain, which is probably the cofactor-binding domain of AHAS (Kim et al 2004) deletion of which affects the AHAS activity. Interestingly, the proposed ''core catalytic region'' of MIPS also resides in the C terminal region (Majumder et al 2003).…”

Section: Discussionmentioning

confidence: 99%

“…The active fractions were also found to be equally crossreactive, like a positive control against the anti-MIPS antibody at 1:1,000 dilution. Although the sll1981 has been putatively annotated as ALS, attempts to assay the purified bacterially expressed sll1981 protein for ALS activity by the method of Kim et al (2004) were so far unsuccessful. Multiple sequence alignment of yeast MIPS sequence (GeneBank Prot.…”

Section: Bacterial Overexpression Purification and In Vitro Mips Acmentioning

confidence: 99%

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

Chatterjee

Dastidar

Maitra

et al. 2006

Planta

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L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of D-glucose 6-phosphate to L-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989-998, 2004). One of the forms with an approximately 50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as an approximately 65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified approximately 1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive approximately 65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.

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“…The residues between 580 and 595 were disordered in the crystal structure of the free CSU of yeast,9b whereas the equivalent residues formed a helix in the A. thaliana crystal structure with inhibitor 14b. 29 The corresponding residues in ilvI (residues 478–493) were modeled as a helix in light of the many reports on the importance of this region for biological function: 1) the well‐conserved region in AHAS;1c 2) participation in the binding/stabilization of the active dimer of CSU and ThDP binding;30 3) its influence on the conformation of ThDP;2a 4) participation in the formation of a substrate access channel;29 5) involvement in substrate specificity (Val395, Phe113, Met480 and Trp484 in E. coli AHAS III)31 and herbicides binding (Met480, Val481 and Trp484 in E. coli AHAS III);32 and 6) ordered upon herbicide binding 2a. The structure of the ilvH dimer was rendered from E. coli AHAS III ilvH crystal structure (PDB ID: 2F1F).…”

Section: Discussionmentioning

confidence: 99%

Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

et al. 2012

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Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein-protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins.

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

Cited by 16 publications

References 41 publications

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

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