Purification, Characterization and Molecular Studies of Fructose-6-Phosphate Phosphoketolase (F6PPK) from Bifidobacteria

Fructose-6-phosphate phosphoketolase (F6PPK; EC 4.1.2.22) is the key enzyme in the fructose-6-phosphate shunt pathway of glucose metabolism which is apparently restricted to bifidobacteria. Despite the biological importance of this bacterial group and the heterogeneity of the enzyme from different species, F6PPK in itself has not been characterized in detail with respect to size, subunit number, steady kinetics and N-terminal sequence. F6PPK was extracted and characterized for the first time from Bifidobacterium asteroides (isolated from the intestine of honeybees; ATTC 25909). The enzyme was purified to homogeneity using acetone fractionation at 40-70% saturation followed by fast protein liquid chromatography (FPLC) on Mono-Q anion exchange and Superose 12 gel filtration columns. The intact enzyme has a relative molecular mass of 110 ± 5 kDa as estimated by gel filtration chromatography (Sephadex G-200), and a single band was obtained on nondenaturingP AGE. It was then shown to be that of F6PPK following elution from preparative polyacrylamid gel. Sodium dodecyl sulphate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 110 ± 2 kDa. SDS-PAGE of F6PPK reacted with 2-mecaptoethanol revealed the presence of two polypeptides of 59 ± 1 and 53 ± 0.5 kDa, indicating a dimeric structure (α₁ β₁) with disulfide-linked subunits. The NH2-terminal amino acid of the a. chain was found to be methionine. The enzyme was stable at pH 4.5-8.0 with an optimum activity at pH 6.0. The enzyme was stable below 42°C and the optimum temperature was 30°C. The apparent Km value of the enzyme for fructose-6-phosphate was 14.1 mM. The purified enzyme has no apparent requirement for thiamine pyrophosphate as cofactors. The enzyme was inactivated by Hg2+ and recovered after addition of dithiothretol, indicating that sulfhydryl group was probably involved in the enzyme activity. The features of B. asteroides F6PPK showed marked differences from those previously reported from animal and human strains. F6PPK from Bifidobacterium longum (probiotic grade; BB536) was also purified to electrophoretic homogeneity using the same purification steps above. The purified enzyme had a molecular mass of about 300 kDa as determined by gel filtration on Superose 12. F6PPK migrated as a single electrophoretic band in nondenaturing polyacrylamide gel electrophoresis (PAGE). It is probably a tetramer containing two different subunits with molecular masses of about 93 ± 1 kDa and 59 ± 0.5 kDa, as determined by SDS-PAGE. The N-terminal amino acid sequences of the subunits were determined, and no significant similarity was found between the deduced amino acid sequences and those in the databases of EMBL and SWISSPORT, indicating that we may be reporting for the first time the partial sequence of F6PPK from two type strains of Bifidobacterium species. However, the Mr 59000 subunit of B. asteroides F6PPK showed a significant similarity (70%) with the corresponding subunit from B. longum species. Oligonucleotide probes which were designed based on the deduced N-terminal amino acids sequences were unable to detect the presence of F6PPK gene using dot blot and Southern blot of the total genomic DNA from different species of bifidobacteria and other bacterial strains. In addition, the genomic library of B. asteroides was constructed in BamHI-digested pUCI9 by using about 2 to 6-kb DNA fragments obtained by partial digestion of the total genomic DNA with BamHI. The transformed cells efficiency of E. coli XLI- blue carrying plasmids with genomic inserts was 1.1 x 104 cfu mrl, and this library may be a useful tool for fishing the gene encoding for F6PPK

P53 Codon 72 Polymorphism and Risk for Squamous Cell Carcinoma of the Penis: A Caucasian Case-Control Study

Squamous cell carcinoma of the penis is a rare but often aggressive disease. A large proportion of penile cancers are associated with HPV infection, mainly with HPV high-risk subtypes 16 and 18. From other HPV-related malignancies a link between a functional SNP in the p53 gene (rs1042522, p.Arg72Pro) and a higher disease risk in the presence of HPV is documented. The p53 p.Arg72 variant was described as a risk factor for developing a malignancy in combination with the presence of HPV as the p.72Arg variant is more prone to HPV E6 protein-mediated degradation than the p.72Pro variant. For penile carcinoma there are only sparse data available on this topic. We therefore analyzed the distribution of this p53 codon 72 SNP in a cohort of 107 penile cancer patients and a healthy control group (n=194) using Restriction Fragment Length Polymorphism (RFLP) analysis. After DNA isolation a PCR amplicon including the variant nucleotide was generated. Based on the variant nucleotide this amplicon can be cleaved into two parts or remain unaffected by a restriction enzyme. Subsequent electrophoresis allowed the discrimination of SNP alleles in the investigated sample. Comparison of the allelic variants revealed no significant differences in the distribution of this SNP between cases and controls (p=0,622). There was also no difference in SNP distribution between cases with/without HPV infection (p=0,558) or histologic variants (p=0.339). In order to strengthen the impact of our data we performed a combined analysis of all published data on this topic with our results. This ended up in SNP distribution data from 177 cases and 1149 controls. Overall, there were also no significant differences in the allelic distribution of the p53 codon 72 SNP between either cases and controls (p=0,914) or HPV-positive and HPV-negative cases (p=0,486). From this most comprehensive data available to date we conclude that there is no influence of the p53 codon 72 SNP on the risk of development of penile carcinoma in Caucasians even in the presence of HPV