Characterization and structure of glyceraldehyde-3-phosphate dehydrogenase type 1 from Escherichia coli.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway that catalyzes the conversion of D-glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. Here, the full-length GAPDH type 1 from Escherichia coli (EcGAPDH1) was cloned and overexpressed, and the protein was purified. Biochemical analyses found that the optimum reaction temperature and pH of EcGAPDH1 were 55°C and 10.0, respectively. The protein has a certain amount of thermostability. Crystals of EcGAPDH1 were obtained using the sitting-drop vapor-diffusion technique and X-ray diffraction data were collected to 1.88 Šresolution. Characterization of the crystals showed that they belonged to space group P41212, with unit-cell parameters a = b = 89.651, c = 341.007 Å, α = ß = γ = 90°. The structure of EcGAPDH1 contains four subunits, each of which includes an N-terminal NAD+-binding domain and a C-terminal catalytic domain. Analysis of the NAD+-bound form showed some differences between the structures of EcGAPDH1 and human GAPDH. As EcGAPDH1 shares 100% identity with GAPDH from Shigella sonnei, its structure may help in finding a drug for the treatment of shigellosis.

Cloning and expression of ARMC3_v2, a novel splicing variant of the human ARMC3 gene.

ARM genes, whose polypeptide consist of Armadillo/beta-catenin-like repeats (ARM) domain(s), exist ubiquitously from fly to vertebrates. These genes have multiple functions in signal transduction, development, cell adhesion and mobility, tumor initiation and metastasis. In this study, we have isolated a novel splicing variant of ARMC3 from human fetal brain, which is 2439 bp, encoding a 688-amino acid polypeptide that contains three typical ARM domains. The cDNA called ARMC3_v2 and the original called ARMC3_v1 (GeneBank: BC039312) are both located on the human chromosome 10p12.23. RT-PCR analysis in our work showed that ARMC3_v2 was detected in human skeletal muscle, liver, spleen and thymus; in contrast, ARMC3_v1 in skeletal muscle, lung, prostate and testis.

[Effects of Pro229-->Ser and Glu243-->Gly on the characters of thermostable catechol 2, 3-dioxygenase].

In order to investigate the effects of amino acid replacement on the characters of thermostable catechol 2, 3-dioxygenase, two mutants (Pro229Ser and Glu243Gly) of this enzyme were obtained by using the method of PCR random mutagenesis. The wild type thermostable catechol 2, 3-dioxygenase and these two mutants (Pro229Ser, Glu243Gly) were over expressed in E. coli TG1 and purified. The enzymatic characters and thermostability of the wild type enzyme and the two mutants (Pro229Ser, Glu243Gly) were analyzed. The results revealed that the optimum enzymatic temperature of the two mutants were the same as that of the wild type enzyme (60 degrees C) and the Kcat/Km value of Pro229Ser and Glu243Gly (4.89 +/- 0.01 x 10(6) mol-1 s-1 and 5.88 +/- 0.01 x 10(6) mol-1 s-1, respectively) were reduced compared with the wild type enzyme (6.97 +/- 0.01 x 10(6) mol-1 s-1). However, the thermostability of Pro229Ser extremely decreased 10.2 degrees C and the thermostability of Glu243Gly slightly increased 1.5 degrees C. It was proposed that Pro229 played an important role on the thermostability of thermostable catechol 2, 3-dioxygenase.

Purification, crystallization and preliminary X-ray studies of thermostable alkaline phosphatase from Thermus sp. 3041.

Thermostable alkaline phosphatase from Thermus sp. 3041 has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2(1)22(1), with unit-cell parameters a = 57.7, b = 69.9, c = 111.5 A. Diffraction data were collected to 2.54 A with a completeness of 91.1% (87.8% for the last shell), an R(merge) value of 0.105 (0.312) and an I/sigma(I) value of 9.5 (3.6).

[A study of a mutant of thermostable alkaline phosphatase].

To reveal the mechanism of protein thermostability, we used in vivo random mutagenesis to generate variants of pTAP503F which contained thermostable alkaline phosphatase (FD-TAP). After screening about 5,000 clones, we obtained 4 temperature-sensitive mutants. The study of enzymatic properties of one mutant (TAPM3) showed that the thermostability of the mutant enzyme descended a lot, compared to the wild type, while the thermoactivity remained stable. DNA sequencing showed that the G-A transition in position 1,239 resulted in the substitution from glysine to serine in position 427. This mutation conspicuously affected thermostability, Michaelis constant and energy of activation. This suggests that only one substitution of amino acid will make great changes in thermostability and other properties, meanwhile, side-chain size, charge of residues and so on, which loosen the structure of protein, will result in the descent of thermostability.

[Studies of the active site, thermostability and thermophilicity of the thermostable alkaline phosphatase by site-directed mutagenesis].

Through PCR-mediated mutagenesis, three mutants E68S, S70A and E68SS70A around active site S(69) were obtained. Their enzymatic characteristics was determined. It was found that the specific activity of E68S ascended 8 times while its optional reactive temperature climbed 20 degrees C and its Tm descended 3 degrees C; the specific activity of S70A ascended 1 time while its optional reactive temperature climbed 5 degrees C and its Tm descended 2 degrees C; the specific activity of E68SS70A descended 50% while its optional reactive temperature climbed 5 degrees C and its Tm descended 19 degrees C. These result implied that the amino acids, beside the active site, were contributed not only to enzymatic activity but also to its thermostability and thermophilicity. The work provided the direction for mutation to improve enzymatic specific activity and studying the mechanism of thermostability and thermophilicity.

[A study of a derivative of Taq DNA polymerase].

Using the method of double primer oligonucleotide-mediated mutagenesis, the high expression plasmid of TaqND236, a derivative of Taq DNA polymerase, was constructed. To determine the frameshift mutation frequency of the in vitro DNA synthesis, we constructed a Gapped-DNA system using the pFDPM118 (a mutant of pUC118 with a -1 frameshift mutation on the lacZ gene) as template. By calculating the ratio of blue and white colonies on the X-gal plate after transforming E. coli TG1, the frameshift mutation frequency of Taq and TaqND236 was measured. It was found that the replication fidelity of the deleted Taq-TaqND236 increased more than 10 folds.