Structural Genomics of Minimal Organisms: Pipeline and Results

Kim, Sung Hou; Shin, Dongkun; Kim, Rosalind; Adams, Paul D.; Chandonia, John-Marc

doi:10.1007/978-1-60327-058-8_32

Cited by 3 publications

(3 citation statements)

References 47 publications

(37 reference statements)

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“…Efforts to determine the structures of all proteins from a microorganism, while not yielding completeness, have nevertheless given a picture of the range of structures involved in supporting a living organism (13, 31, 34, 35, 54) (http://www.thermus.org/e_index.htm). More importantly, they form a foundation for future efforts to develop detailed models of each step in metabolism, regulation, and other cellular functions.…”

Section: Value Of Structures From Structural Genomicsmentioning

confidence: 99%

Lessons from Structural Genomics

Terwilliger

Stuart²,

Yokoyama³

2009

Annu. Rev. Biophys.

120

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A decade of structural genomics, the large-scale determination of protein structures, has generated a wealth of data and many important lessons for structural biology and for future large-scale projects. These lessons include a confirmation that it is possible to construct large-scale facilities that can determine the structures of a hundred or more proteins per year, that these structures can be of high quality, and that these structures can have an important impact. Technology development has played a critical role in structural genomics, the difficulties at each step of determining a structure of a particular protein can be quantified, and validation of technologies is nearly as important as the technologies themselves. Finally, rapid deposition of data in public databases has increased the impact and usefulness of the data and international cooperation has advanced the field and improved data sharing.

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Section: Value Of Structures From Structural Genomicsmentioning

confidence: 99%

Lessons from Structural Genomics

Terwilliger

Stuart²,

Yokoyama³

2009

Annu. Rev. Biophys.

120

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“…Initially, PamH was mainly expressed in inclusion bodies with 0.1 mM IPTG induction, either at 37°C or 18°C in LB media. However, after we changed the LB media to autoinduction media (Studier 2005;Kim et al 2008), approximately 10% of PamH was expressed as the soluble protein (data not shown). Obviously, the rapid expression of PamH caused inefficient protein folding.…”

Section: Discussionmentioning

confidence: 97%

“…The positive transformants were selected and grown in 1 L of auto-inducing media ZYM-5052 (Studier 2005;Kim et al 2008) with kanamycin (100 mg l −1 ) for 16 h at 25°C and with shaking at 200 rpm. The cells were harvested, washed, and resuspended in Tris-HCl buffer (10 mM, pH 8.0) at 4°C and lysed by sonication on ice.…”

Section: Expression and Purification Of Amidasementioning

confidence: 99%

Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities

Shen

Chen

Jia

et al. 2011

Appl Microbiol Biotechnol

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A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45°C and at pH 8.0 and was stable within a pH range of 5.0-10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the k (cat) and K (m) were 2.8 s(-1) and 158 μM, respectively, and the catalytic efficiency value (k (cat)/K (m)) was 0.018 μM(-1) s(-1). In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.

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Molecular Characterization of a Novel Bacterial Aryl Acylamidase Belonging to the Amidase Signature Enzyme Family

Lee

Bang

et al. 2010

Molecules and Cells

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In seeking aryl acylamidase (EC 3.5.1.13) acting on an amide bond in p-acetaminophenol (Tylenol), we identified a novel gene encoding 496 residues of a protein. The gene revealed a conserved amidase signature region with a canonical catalytic triad. The gene was expressed in E. coli and characterized for its biochemical properties. The optimum pH and temperature for the activity on p-acetaminophenol were 10 and 37 degrees C, respectively. The half-life of enzyme activity at 37 degrees C was 192 h and 90% of its activity remained after 3 h incubation at 40 degrees C. Divalent metals was found to inhibit the activity of enzyme. The K (m) values for various aryl acylamides such as 4-nitroacetanilide, p-acetaminophenol, phenacetin, 4-chloroacetanilide and acetanilide were 0.10, 0.32, 0.83, 1.9 and 19 mM, respectively. The reverse reaction activity (amide synthesis) was also examined using various chain lengths (C(1) approximately C(4) and C(10)) of carboxylic donors and aniline as substrates. These kinetic parameters and substrate specificity in forward and reverse reaction indicated that the aryl acylamidase in this study has a preference for aryl substrate having polar functional groups and hydrophobic carboxylic donors.

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Structural Genomics of Minimal Organisms: Pipeline and Results

Cited by 3 publications

References 47 publications

Lessons from Structural Genomics

Lessons from Structural Genomics

Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities

Molecular Characterization of a Novel Bacterial Aryl Acylamidase Belonging to the Amidase Signature Enzyme Family

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