Pyrazinamide (PZA) is one of the most important drugs for the treatment of Mycobacterium tuberculosis infection. However, the increasing frequency of PZA-resistant strains limits its effectiveness. In Korea, most PZA-resistant strains also exhibit both isoniazid and rifampin resistance making it essential to identify these resistant strains accurately and rapidly for effective treatment of mycobacterial infection. In this study, the characteristics and frequency of mutations of the pncA gene encoding pyrazinamidase were investigated in PZA-resistant clinical isolates from Korea. Automated DNA sequencing was used to evaluate the usefulness of DNA-based detection of PZA resistance. Among 95 PZA-resistant clinical isolates, 92 (97%) exhibited mutations potentially affecting either the production or the activity of the enzyme. Mutations were found throughout the pncA gene including the upstream region. Single nucleotide replacement appeared to be the major mutational event (69/92), although multiple substitutions as well as insertion and deletion of nucleotides were also identified. The high frequency of pncA mutations observed in this study supports the usefulness of DNA-based detection of PZA-resistant M. tuberculosis. Having verified the scattered and diverse mutational characteristics of the pncA gene, automated DNA sequencing seems to be the best strategy for rapid detection of PZA-resistant M. tuberculosis.
Amidohydrolases/*genetics ; Antitubercular Agents/*pharmacology ; Drug Resistance, Bacterial ; *Mutation ; Mycobacterium tuberculosis/*drug effects/genetics ; Pyrazinamide/*pharmacology

The amidase of Nocardia sp. is one of important industrial enzymes. Based on DNA and protein sequence alignment from different strains, a new gene of amidase was successfully cloned from Nocardia YS-2002, which is widely used for industrial production of acrylamide in China. DNA sequence analyses showed that the 1466bp cloned-fragment contains promoter, open reading frame and terminating-palindrome. Protein sequence alignment and phylogenetic tree analyses showed that the amidase coming from Nocardia sp. YS-2002 is a kind of specialamidase, without the typical conserved sequence of the amidases. Enzymatic characteristics predictions indicated that the molecular weight and pI of the new amidase is approximately 38.05 kD and 4.88, respectively, and it would be stable when heterogeneously expressed in E. coli. By inserting the ORF of the amidase into plasmid pET-28a(+), a recombinant strain, pEAB, was selected using E. coli BL21(DE3) as the host. SDS-PAGE analyses of both the whole cells and ultrasonic-treated cells confirmed the feasibility of the heterogeneous expression of amidase in the recombinant E. coli. But the activity of amidase in E. coli BL21(DE3) not more than 0.5 u/mg, because most of the enzymes expressed were formed as inclusion bodies.
Amidohydrolases ; chemistry ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; Molecular Weight ; Nocardia ; enzymology ; Phylogeny

OBJECTIVE: To analyze the molecular etiology of a Chinese child affected with dihydropyrimidinase deficiency. METHODS: Genomic DNA was extracted from peripheral blood samples of the family members. Pathogenic variant was determined by whole exome sequencing and verified by Sanger sequencing. RESULTS: The child was found to harbor homozygous c.905G>A (p.Arg302Gln) variants in exon 5 of the DPYS gene, for which her parents were both heterozygous carriers. CONCLUSION The homozygous c.905G>A (p.Arg302Gln) variants of the DPYS gene probably underlies the dihydropyrimidinase deficiency in the child. Above result has enabled genetic counseling and prenatal diagnosis for this family.
Amidohydrolases/genetics* ; Asian Continental Ancestry Group/genetics* ; Child ; Exons ; Female ; Humans ; Metabolism, Inborn Errors/genetics* ; Mutation ; Pedigree

In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.
Amidohydrolases ; genetics ; metabolism ; Bile Acids and Salts ; metabolism ; Escherichia coli ; metabolism ; Gene Expression ; Lactobacillus ; enzymology ; genetics ; Substrate Specificity

The present study analyzed the correlations between curcumin(Cur), nuclear factor E2 related factor 2(NRF2)-dimethylarginine dimethylaminohydrolase(DDAH)-asymmetric dimethylarginine(ADMA)-nitric oxide(NO) pathway, and endothelial-mesenchymal transition(EndMT) based on SD rats with cardiac fibrosis, and explored the effect and mechanism of Cur in resisting cardiac fibrosis to provide an in-depth theoretical basis for its clinical application in the treatment of heart failure. The cardiac fibrosis model was induced by subcutaneous injection of isoprenaline(Iso) in rats. Thirty-two rats were randomly divided into a control group, a model group, a low-dose Cur group(100 mg·kg~(-1)·d~(-1)), and a high-dose Cur group(200 mg·kg~(-1)·d~(-1)), with eight in each group. After 21 days of treatment, cardiac function was detected by echocardiography, degree of cardiac fibrosis by Masson staining, expression of CD31 and α-SMA by pathological staining, expression of VE-cadherin, vimentin, NRF2, and DDAH by Western blot, and ADMA level by HPLC. Compared with the model group, the Cur groups showed alleviated cardiac fibrosis, accompanied by increased CD31 and VE-cadherin expression and decreased α-SMA and vimentin expression, indicating relieved EndMT. Additionally, DDAH and NRF2 levels were elevated and ADMA and NO expression declined. Cur improves cardiac fibrosis by inhibiting EndMT presumedly through the NRF2-DDAH-ADMA-NO pathway.
Amidohydrolases/metabolism* ; Animals ; Curcumin ; Fibrosis ; NF-E2-Related Factor 2/genetics* ; Nitric Oxide/metabolism* ; Rats ; Rats, Sprague-Dawley

Hydantoin-utility-enzyme is widely used in enzymic production of various amino acids. One of its component, carbamoylase, is responsible for the conversion of N-carbamylamino acids to corresponding amino acids, which is crucial for the stereoselectivity and rate limiting. To improve the production of the enzyme, an L-N-carbamoylase gene from Arthrobacter BT801, a hydantoinase producting strain being able to convert 5-benzylhydantoin to phenylalanine, was cloned into E. coli. The gene was highly expressed in E. coli M15 under control of T5 promoter. A protein band about 44kD was detected by SDS-PAGE in the recombinant cell lysate. The objective product, which is principally in soluble form, represented 40% of total cell protein. The N-carbamoylase specific activity of the recombinant M15/pQE60- hyuC is 53 times higher than that of Arthrobacter BT801. The total biotransformation activity increased 8.1 times when. M15/pQE60-hyuC was added into the Arthrobacter BT801 reaction system. The successful expression of the enzyme is significant for the application of the hydantoinase producing strain or the enzyme thereof.
Amidohydrolases ; genetics ; metabolism ; Arthrobacter ; enzymology ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hydantoins ; metabolism ; Models, Genetic ; Phenylalanine ; metabolism ; Plasmids ; genetics ; Polymerase Chain Reaction

Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.
Amidohydrolases ; genetics ; Atrazine ; metabolism ; Bacterial Proteins ; genetics ; Biodegradation, Environmental ; Chlorophyta ; genetics ; metabolism ; Herbicides ; metabolism ; High-Throughput Screening Assays ; Hydrolases ; biosynthesis ; genetics ; Mutagenesis, Insertional ; Pseudomonas ; enzymology ; genetics

OBJECTIVE To detect potential mutation in a Chinese family affected with beta-ureidopropinoase deficiency. METHODS Genomic DNA was extracted from peripheral blood samples. All exons and flanking intron regions of the UPB1 gene were amplified by PCR and detected by direct sequencing. RESULTS A homozygous mutation c.977G>A was identified in exon 9 of the UPB1 gene in the proband. Both parents of the proband had heterozygous change of the same site. CONCLUSION The c.977G>A mutation of the UPB1 gene is responsible for the pathogenesis of the disease in the infant.
Abnormalities, Multiple ; genetics ; Amidohydrolases ; deficiency ; genetics ; Brain Diseases ; genetics ; Exons ; Humans ; Infant ; Male ; Movement Disorders ; genetics ; Mutation ; Purine-Pyrimidine Metabolism, Inborn Errors ; genetics

OBJECTIVETo assess the association of polymorphisms of N-acyl-phosphatidylethanolamine-phospholipase D (DAPE-PLD) and fatty acid amide hydrolase (FAAH) genes, as well as their interaction, with schizophrenia.

METHODSPolymorphisms of NAPE-PLD rs12540583 and FAAH rs324420, rs2295633, and rs6429600 were determined with PCR - restriction fragment length polymorphism assay and Sanger sequencing. The genotypes of 345 subjects of Han Chinese origin diagnosed with schizophrenia and a 403 controls were compared. The results were analyzed with SPSS 17.0, and the interaction of the two genes was analyzed using a multifactor dimensionality reduction (MDR) method.

RESULTSThe frequency of NAPE-PLD rs12540583 polymorphism was significantly different between the two groups under both dominant and additive models (χ2=17.18 vs. χ2=18.94, P<0.0125). The frequencies of AC genotype and C allele of the patient group at rs12540583 were higher than those of the controls, and the interaction of NAPE-PLD and FAAH was associated with schizophrenia. A four-loci model (rs12540583, rs324420, rs2295633 and rs6429600) can best model the interaction between NAPE-PLD and FAAH.

CONCLUSIONThe AC genotype and C allele of NAPE-PLD rs12540583 locus are risk factors for schizophrenia, and the interaction between NAPE-PLD rs12540583 and FAAH rs324420, rs2295633 and rs6429600 is associated with schizophrenia.

Adult ; Amidohydrolases ; genetics ; Asian Continental Ancestry Group ; genetics ; China ; ethnology ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Phospholipase D ; genetics ; Polymorphism, Genetic ; Schizophrenia ; genetics

Ethyl carbamate as a potential carcinogen commonly exists in traditional fermented foods and beverages. Enzymatic removal of ethyl carbamate from fermented foods and beverages is an efficient and safe method. In this study, we mutated urethanase from Lysinibacillus fusiformis SC02 on the Q328 site through computer aided design approaches. The half-life of resulting mutants Q328C and Q328V was detected to be 7.46 and 1.96 folds higher than that of the original enzyme, and Q328R presented better thermal-tolerance than the original urethanase when incubated at high temperature. The tolerance of Q328C to ethanol and acid also increased when compared with that of the original enzyme. The stability and tolerance to acid and ethanol of urethanase could be improved by modification on its Q328 site.
Amidohydrolases ; biosynthesis ; genetics ; Bacillaceae ; enzymology ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Computer-Aided Design ; Enzyme Stability ; Ethanol ; Mutagenesis, Site-Directed ; Protein Engineering