Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two isozymes of arylamine N-acetyltransferases (NATs) catalyze the biotransformation of arylamines to arylamides, and the bioactivation of carcinogenic arylhydroxylamines and arylhydroxamic acids to reactive electrophiles capable of forming a variety of DNA and protein adducts. As part of a project directed toward delineation of the molecular factors responsible for the pronounced differences in the substrate specificity of the isozymes, we have recently reported the expression in Escherichia coli and purification of hamster NAT1 (NAT1 8) as a fusion protein to an antibody-reactive amino terminus FLAG peptide capable of being removed by digestion with enterokinase. Unfortunately, the conditions necessary for the removal of the peptide by enterokinase resulted in incomplete protease digestion and substantial loss of NAT1 activity. Consequently, we have constructed the plasmid pPH8 in which an 11 amino acid thrombin proteolysis site has been inserted between the FLAG peptide and the amino terminus of NAT1. In addition, a plasmid that expresses hamster NAT2 (NAT2 15) was constructed by exchanging the gene sequence for NAT1 with the cloned sequence for NAT2. Both NAT fusion proteins were expressed in JM105 cells. Analysis of catalytically active cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that approximately 30-fold more soluble NAT2 was expressed than NAT1 in the bacterial cytosol. The fusion proteins were purified to homogeneity by immunoaffinity chromatography, followed by gel filtration to remove high molecular weight contaminants. The FLAG peptide was subsequently removed by treatment with human thrombin, followed by an additional pass over the immunoaffinity column. Unlike the results obtained from proteolysis by enterokinase, the activities of the rNAT1 and rNAT2 were shown to be unaffected by treatment with thrombin. Moreover, the substrate specificities for the recombinant NATs closely matched those observed for NAT1 and NAT2 isolated from hamster liver.
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PMID:Arylamine N-acetyltransferases. Expression in Escherichia coli, purification, and substrate specificities of recombinant hamster monomorphic and polymorphic isozymes. 874 38

Arylamine N-acetyltransferase (NAT) acetylates the front-line anti-tuberculosis drug isoniazid (INH) and has been identified in Mycobacterium tuberculosis. A naturally occurring single nucleotide polymorphism (SNP) was recently found in the NAT gene in clinical isolates of M. tuberculosis. The nucleotide change from G-->A (619) produces an amino acid change Gly(207) Arg, which appears to reduce the activity of the NAT from M. tuberculosis (TBNAT). It has not been possible to generate sufficient soluble recombinant TBNAT for 3D structural studies. Therefore, Mycobacterium smegmatis NAT (SMNAT), which has 60% identity to TBNAT and has Gly at 207, was used as a model to investigate the possible structural effects of the G-->A 619 SNP. The mutant form of SMnat (SM207Rnat) was constructed by in vitro site-directed mutagenesis and was heterologously expressed with an N-terminal His tag in Escherichia coli, for comparison with the SMNAT. Both recombinant SMNATs were purified using Ni affinity chromatography and treated with thrombin to cleave the tag. Both proteins were produced with average yields of over 10 mg/L and were active. Substrate specificity and thermal stability of SM207RNAT were assessed and compared with the wild type SMNAT using kinetic assays and circular dichroism spectroscopy. SM207RNAT was crystallised and a data set of 2.00 A resolution was obtained. The SM207RNAT had different substrate specificities to the wild type protein and the 3D structures revealed that the Gly(207) Arg mutation caused slight changes in the orientation of His(203) in SMNAT.
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PMID:Structural investigation of mutant Mycobacterium smegmatis arylamine N-acetyltransferase: a model for a naturally occurring functional polymorphism in Mycobacterium tuberculosis arylamine N-acetyltransferase. 1250 87