• Bulletin of the Korean Chemical Society
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• 제23권5호
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• pp.765-768
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• 2002

• Bulletin of the Korean Chemical Society
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• 제24권12호
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• pp.1856-1858
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• 2003

• Bulletin of the Korean Chemical Society
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• 제24권5호
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• pp.627-632
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• 2003

The pH dependence of the kinetic parameters of recombinant acetohydroxy acid synthase catalyzed reaction was determined in order to obtain information about the chemical mechanism, particularly acid-base chemistry. The maximum velocity and V/K for pyruvate were bell-shaped with estimated pK values of 6.5-6.7 and 8.6-8.9, respectively. The maximum velocity and V/K for 2-ketobutyrate were also bell-shaped with estimated pK values of 6.6-7.0 and 8.4-8.6. The pH dependence of 1/Ki for 3-bromopyruvate, a competitive inhibitor of pyruvate, was also bell-shaped, giving pK values almost identical with those obtained for pyruvate. Since the same pK values were observed in the $pK_{i 3-bromopyruvate}$, V/K pH profiles and $V_{max}$ profiles, both enzyme groups must be in their optimum protonation state for efficient binding of reactants. These results reflect that two enzyme groups are necessary for binding of substrate and/or catalysis.

• Bulletin of the Korean Chemical Society
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• 제26권6호
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• pp.916-920
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• 2005

Acetohydroxy acid synthase catalyzes the first common step in the biosynthesis of branched chain amino acids. AHAS plays two distinct metabolic roles, and is designated as anabolic AHAS and catabolic AHAS, depending on its function. Anabolic AHAS is FAD-dependent, while its catabolic counterpart is not. In this work, a conserved motif was identified in the $\beta$-domain of anabolic AHASs, but not in catabolic AHAS ($_{372}RFDDR_{376}$). In order to determine the functions of this motif, we replaced the motif with the corresponding sequence in FAD-independent AHAS, SPVEY. None of these three mutants (SPV, SPVE, and SPVEY) was detected with bound FAD. However, two of these mutants (SPVE and SPVEY) were active at a low level of specific activity. Although they exhibited pyruvate- and ThDP- dependent characteristics, the activity of the two active mutants appears to be FAD-independent. The SPVEY mutant was completely insensitive to the three tested herbicides, even at extremely high concentrations and is also somewhat more thermolabile than the wild type enzyme. The data provided in this work suggest that the RFDDR motif is a possible determinant of the FAD-dependent and herbicide-resistant properties of tobacco AHAS. The SPVEY mutant appears to exhibit catabolic AHAS-like activity.

• Bulletin of the Korean Chemical Society
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• 제28권6호
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• pp.947-952
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• 2007

Mycobacterium tuberculosis is a pathogen responsible for 2-3 million deaths every year worldwide. The emergence of drug-resistant and multidrug-resistant tuberculosis has increased the need to identify new antituberculosis targets. Acetohydroxy acid synthase, (AHAS, EC 2.2.1.6), an enzyme involved in branched-chain amino acid synthesis, has recently been identified as a potential anti-tuberculosis target. To assist in the search for new inhibitors and “receptor-based” design of effective inhibitors of tubercular AHAS (TbAHAS), we constructed four different structural models of TbAHAS and used one of the models as a target for virtual screening of potential inhibitors. The quality of each model was assessed stereochemically by PROCHECK and found to be reliable. Up to 89% of the amino acid residues in the structural models were located in the most favored regions of the Ramachandran plot, which indicates that the conformation of each residue in the models is good. In the models, residues at the herbicide-binding site were highly conserved across 39 AHAS sequences. The binding mode of TbAHAS with a sulfonylurea herbicide was characterized by 32 hydrophobic interactions, the majority of which were contributed by residue Trp516. The model based on the highest resolution X-ray structure of yeast AHAS was used as the target for virtual screening of a chemical database containing 8300 molecules with a heterocyclic ring. We developed a short list of molecules that were predicted to bind with high scores to TbAHAS in a conformation similar to that of sulfonylurea derivatives. Five sulfonylurea herbicides that were calculated to efficiently bind TbAHAS were experimentally verified and found to inhibit enzyme activity at micromolar concentrations. The data suggest that this time-saving and costeffective computational approach can be used to discover new TbAHAS inhibitors. The list of chemicals studied in this work is supplied to facilitate independent experimental verification of the computational approach.

• Bulletin of the Korean Chemical Society
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• 제25권5호
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• pp.721-725
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• 2004

Acetohydroxy acid synthase (AHAS) is one of several enzymes that require thiamine diphosphate and a divalent cation as essential cofactors. The active site contains several conserved ionizable groups and all of these appear to be important as judged by the fact that mutation diminishes or abolishes catalytic activity. Recently, we have shown [Yoon, M.-Y., Hwang, J.-H., Choi, M.-K., Baek, D.-K., Kim, J., Kim, Y.-T., Choi, J.-D. FEBS Letters 555 (2003), 185-191] that the activity is pH-dependent due to changes in $V_{max}$ and V/$K_m$. Data were consistent with a mechanism in which substrate was selectively catalyzed by the enzyme with an unprotonated base having a pK 6.48, and a protonated group having a pK of 8.25 for catalysis. Here, we have in detail studied the pH dependence of the kinetic parameters of the cofactors (ThDP, FAD, $Mg^{2+}$) in order to obtain information about the chemical mechanism in the active site. The $V_{max}$ of kinetic parameters for all cofactors was pH-dependent on the basic side. The pK of ThDP, FAD and $Mg^{2+}$ was 9.5, 9.3 and 10.1, respectively. The V/$K_m$ of kinetic parameters for all cofactors was pH-dependent on the acidic and on the basic side. The pK of ThDP, FAD and $Mg^{2+}$ was 6.2-6.4 on the acidic side and 9.0-9.1 on the basic side. The well-conserved histidine mutant (H392) did not affect the pH-dependence of the kinetic parameters. The data are discussed in terms of the acid-base chemical mechanism.

• 한국미생물·생명공학회지
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• 제26권1호
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• pp.45-49
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• 1998

Corynebacterium glutamicum ATCC13032를 모균주로 한 아미노산 유사체들에 저항성을 지닌 돌연변이 균주들로부터 두 종류의 L-로이신 생산균주를 개발하였다. 그 중 하나인 C. glutamicum LT26은 4-azaleucine과 $\alpha$-amino-$eta$-hydroxyvaleric acid에 저항성을 지니는 균주이며, 다른 한 균주는 C. glutamicum LT3811-70로서 C. glutamicum LT26을 모균주로한 DL-4-tiaisoleucine 저항성 돌연변이 균주이다. 이들 두 돌연변이 균주들의 배양액내에서의 L-로이신의 축적은 이들이 비영양요구성 균주임에도 불구하고 모균주보다 획기적으로 높았으며 이를 해명하고자 L-이소로이신과 L-발린 그리고 L-로이신 생합성 과정의 주반응 효소인 $\alpha$-acetohydroxy acid synthase(AHAS)와 $\alpha$-isopropylmalate synthase(IPMS)의 분석을 수행하였다. C. glutamicum LT26과 LT3811-70에서 AHAS와 IPMS는 모두 L-로이신에 대해 효소활성저해와 효소합성저해가 거의 해제되었고, C. glutamicum LT3811-70 균주의 경우 모균주인 C. glutamicum LT26 균주보다 IPMS의 L-로이신에 대해 효소 합성저해가, AHAS는 L-이소로이신과 L-발린등에 대해 효소활성저해가 10% 이상 더 해제되었음을 알 수 있었다.

• Weed & Turfgrass Science
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• 제6권1호
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• pp.28-31
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• 2017

The mutation rate of proline in the position 197 (Pro197) in acetohydroxy acid synthase (AHAS) is highest among sulfonylurea (SU) herbicide-resistance mutants. Therefore, it is significant to investigate the resistance mechanism for the mutation and to develop the herbicides specific to the mutants. SU herbicide resistance mechanism of the mutants, 197Ser, 197Thr and 197Ala, in AHAS were targeted for designing new SU-herbicide. We did molecular dynamics (MD) simulation for understanding SU herbicide-resistance mechanisms of AHAS mutants and designed new herbicides with docking and MD evaluations. We have found that mutation to 197Ala and 197Ser enlarged the entrance of the active site, while 197Thr contracted. Map of the root mean square derivation (RMSD) and radius gyrations (Rg) revealed the domain indicating the conformations for herbicide resistant. Based on the enlarging-contracting mechanism of active site entrance, we designed new herbicides with substitution at the heterocyclic moiety of a SU herbicide for the complementary binding to the changed active site entrances of mutants, and designed new herbicides. We confirmed that our screened new herbicides bonded to both AHAS wild type and mutants with higher affinity, showing more stable binding conformation than the existing herbicides.

• Journal of Microbiology and Biotechnology
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• 제7권5호
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• pp.341-344
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• 1997

Batch cultures of Anacystis nidulans BD-1 resistant to azaleucine and fluorotyrosine produced and liberated a wide range of amino acids, notably glutamic acid, alanine, phenylalanine, leucine, isoleucine, cysteine and methionine. Sustained liberation for prolonged periods was achieved after immobilization on calcium alginate and the net concentration in the medium was 0.18-0.2 g $I^{-1}$. While acetohydroxy acid synthase in azaleucine-resistant mutant lost leucine- and isoleucine-sensitivity, fluorotyrosine-resistant strain turned phenylalanine activating. The activities of nitrate assimilating enzymes were also higher in the mutants and were relaxed from ammonium-repression. The metabolic adjustments involved in amino acid overproduction are discussed.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2003년도 제2차 연례학술대회 발표논문집
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• pp.277-287
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• 2003

Acetolactate synthase (ALS, EC 4.1.3.18; also referred to as acetohydroxy acid synthase) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in microorganisms and plants. Recently X-ray structure of yeast ALS was available. Pair-wise alignment of yeast and tobacco ALS sequences revealed 63% sequence similarity. Using Deep View and automatic modeling on Swiss model server, we have generated reliable models of tobacco ALS based on yeast ALS template with a calculated pair-wise RMSD of 0.86 Angstrom. Functional roles of four residues located on the subunit interface (H142, El43, M350, and R376) were examined by site-directed mutagenesis. Seven mutants were generated and purified, of which three mutants (H142T, M350V, and R376F) were found to be inactivated under various assay conditions. The H142k mutant showed moderately altered kinetic properties. The E143A mutant increased 10-fold in K$_m$ value while other parameters remained unchanged. The M350C mutant was strongly resistant to three tested herbicides, while the R376k mutant can bind with herbicide carder at similar affinity to that of wild type enzyme, as determined by tryptophan quenching study. Except M350V mutant, all other mutants were ate to bind with cofactor FAD. Taken together, it is likely that residues H142 and E143 are located at the active site, while residues M350 and R376 are possibly located at the overlapping region of active site and herbicide binding site of the enzyme. Our data also allows us to hypothesize that the interaction between side chains of residues M350 and R376 are probably essential for the correct conformation of the active site. It remains to be elucidated that, whether the herbicide, upon binding with enzyme, inactivates the enzyme by causing change in the active site allosterically, which is unfavorable for catalytic activity.