A continual spectrophotometric assay for amino acid decarboxylases

Scriven, Fiona; Wlasichuk, Kenneth B.; Palcic, Monica M.

doi:10.1016/0003-2697(88)90644-6

Cited by 16 publications

(10 citation statements)

References 13 publications

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“…Yeast ODC and mt DAPDC were assayed as described previously [14, 15] by coupling the carbon dioxide produced from decarboxylation to phosphoenol pyruvate (PEP) carboxylase to give oxalacetate, followed by malate dehydrogenase (MDH) catalyzed reduction. Loss of NADH absorbance at 340 nm was followed on a Kontron UVIKON 9420.…”

Section: Methodsmentioning

confidence: 99%

Analysis of catalytic determinants of diaminopimelate and ornithine decarboxylases using alternate substrates

Fogle

Toney

2011

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Diaminopimelate decarboxylase (DAPDC) and ornithine decarboxylase (ODC) are pyridoxal 5′-phosphate dependent enzymes that are critical to microbial growth and pathogenicity. The latter is the target of drugs that cure African sleeping sickness, while the former is an attractive target for antibacterials. These two enzymes share the (β/α)8 (i.e., TIM barrel) fold with alanine racemase, another pyridoxal 5′-phosphate dependent enzyme critical to bacterial survival. The active site structural homology between DAPDC and ODC is striking even though DAPDC catalyzes the decarboxylation of a D stereocenter with inversion of configuration and ODC catalyzes the decarboxylation of an L stereocenter with retention of configuration. Here, the structural and mechanistic bases of these interesting properties are explored using reactions of alternate substrates with both enzymes. It is concluded that simple binding determinants do not control the observed stereochemical specificities for decarboxylation, and a concerted decarboxylation/proton transfer at Cα of the D stereocenter of diaminopimelate is a possible mechanism for the observed specificity with DAPDC.

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Section: Methodsmentioning

confidence: 99%

Analysis of catalytic determinants of diaminopimelate and ornithine decarboxylases using alternate substrates

Fogle

Toney

2011

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…This result indicates that Hp PDF is really a target of compound 1 and its analog compound 2. In parallel, the inhibitory activities of compounds 1 and 2 to H. pylori DC ( Hp DC) were also measured by using the double‐enzyme coupled assay (Scriven et al 1988; Ray et al 2002). However, compounds 1 and 2 have no inhibitory effect on Hp DC, indicating that Hp DC is not a target of compounds 1 and 2.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant Hp DC was expressed and purified in E. coli system. A double‐enzyme coupled assay was used to determine the inhibitory effect of compounds 1 and 2 (Scriven et al 1988; Ray et al 2002). In this assay, Hp DC activity was estimated by monitoring the decrease of specific absorption at 340 nm caused by NADH.…”

Section: Methodsmentioning

confidence: 99%

Peptide deformylase is a potential target for anti‐Helicobacter pylori drugs: Reverse docking, enzymatic assay, and X‐ray crystallography validation

Jian-Hua

Han

et al. 2006

Protein Science

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Colonization of human stomach by the bacterium Helicobacter pylori is a major causative factor for gastrointestinal illnesses and gastric cancer. However, the discovery of anti-H. pylori agents is a difficult task due to lack of mature protein targets. Therefore, identifying new molecular targets for developing new drugs against H. pylori is obviously necessary. In this study, the in-house potential drug target database (PDTD, http://www.dddc.ac.cn/tarfisdock/) was searched by the reverse docking approach using an active natural product (compound 1) discovered by anti-H. pylori screening as a probe. Homology search revealed that, among the 15 candidates discovered by reverse docking, only diaminopimelate decarboxylase (DC) and peptide deformylase (PDF) have homologous proteins in the genome of H. pylori. Enzymatic assay demonstrated compound 1 and its derivative compound 2 are the potent inhibitors against H. pylori PDF (HpPDF) with IC 50 values of 10.8 and 1.25 mM, respectively. X-ray crystal structures of HpPDF and the complexes of HpPDF with 1 and 2 were determined for the first time, indicating that these two inhibitors bind well with HpPDF binding pocket. All these results indicate that HpPDF is a potential target for screening new anti-H. pylori agents. In addition, compounds 1 and 2 were predicted to bind to HpPDF with relatively high selectivity, suggesting they can be used as leads for developing new anti-H. pylori agents. The results demonstrated that our strategy, reverse docking in conjunction with bioassay and structural biology, is effective and can be used as a complementary approach of functional genomics and chemical biology in target identification.

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“…These have incorporated manometric measurements, whereby the amount of CO 2 evolved from the catalysis of DAP by E. coli DAPDC was measured [9,10]. In addition, the activity of DAPDC from Bacillus sphaericus has been monitored enzymatically by coupling CO 2 formation with NADH oxidation [11]. Similarly, trapping and counting 14 CO 2 evolved from [1,[7][8][9][10][11][12][13][14] C]DAP has been used to assay DAPDC from B. sphaericus, Bacillus subtilis, Triticum vulgaris, and Vicia faba [11e14].…”

Section: Introductionmentioning

confidence: 99%

“…In addition, the activity of DAPDC from Bacillus sphaericus has been monitored enzymatically by coupling CO 2 formation with NADH oxidation [11]. Similarly, trapping and counting 14 CO 2 evolved from [1,[7][8][9][10][11][12][13][14] C]DAP has been used to assay DAPDC from B. sphaericus, Bacillus subtilis, Triticum vulgaris, and Vicia faba [11e14]. The amount of L-lysine produced has also been quantified colorimetrically, with a modified ninhydrin reaction from catalysis of DAP by Lemna perpusilla DAPDC [15] or through the turnover of L-lysine via transamination by L-lysine:aketoglutarate ε-aminotransferase [16].…”

Section: Introductionmentioning

confidence: 99%

An optimized coupled assay for quantifying diaminopimelate decarboxylase activity

Peverelli

Perugini

2015

Biochimie

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A continual spectrophotometric assay for amino acid decarboxylases

Cited by 16 publications

References 13 publications

Analysis of catalytic determinants of diaminopimelate and ornithine decarboxylases using alternate substrates

Analysis of catalytic determinants of diaminopimelate and ornithine decarboxylases using alternate substrates

Peptide deformylase is a potential target for anti‐Helicobacter pylori drugs: Reverse docking, enzymatic assay, and X‐ray crystallography validation

An optimized coupled assay for quantifying diaminopimelate decarboxylase activity

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