Pyridoxal kinase （PLK） （EC 2.7.1.35） catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5＇-phosphate （PLP）, an important cofactor for many enzymatic reactions. Bombyx mori, similar t...Pyridoxal kinase （PLK） （EC 2.7.1.35） catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5＇-phosphate （PLP）, an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method （GenBank accession number： DQ452397）. The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 k.Da. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.展开更多
Colorectal cancer remains the third most common cancer in both women and men worldwide.Identifying modifiable dietary factors is crucial in developing primary prevention strategies.Vitamin B6 is involved in more than ...Colorectal cancer remains the third most common cancer in both women and men worldwide.Identifying modifiable dietary factors is crucial in developing primary prevention strategies.Vitamin B6 is involved in more than 100 coenzyme reactions,and may influence colorectal cancer risk in multiple ways including through its role in one-carbon metabolism related DNA synthesis and methylation and by reducing inflammation,cell proliferation,and oxidative stress.Observational studies of dietary or dietary plus supplementary intake of vitamin B6 and colorectal cancer risk have been inconsistent with most studies reporting nonsignificant positive or inverse associations.However,published studies of plasma pyridoxal 5'-phosphate(the active form of vitamin B6) levels consistently support an approximately 30%-50% reduction in risk of colorectal cancer comparing high with low concentrations.The reasons for the discrepancy in the results between dietary-based and plasma-based studies remain unresolved.Other unresolved questions include the effects of vitamin B6 intake in early life(i.e.,childhood or adolescence) and of suboptimal vitamin B6 status on colorectal cancer risk,whether the associations with vitamin B6 differ across molecular subtypes of colorectal cancer,and whether the vitamin B6-colorectal cancer association is modified by genetic variants of one-carbon metabolism.展开更多
The gene iscS-3 from ,4cidithiobacillus ferrooxidans may play a central role in the delivery of sulfur to a variety of metabolic pathways in this organism. For insight into the sulfur metabolic mechanism of the bacter...The gene iscS-3 from ,4cidithiobacillus ferrooxidans may play a central role in the delivery of sulfur to a variety of metabolic pathways in this organism. For insight into the sulfur metabolic mechanism of the bacteria, an integral three-dimensional （3D） molecular structure of the protein encoded by this gene was built by homology modeling techniques, refined by molecular dynamics simulations, assessed by PROFILE-3D and PROSTAT programs and further used to search bind sites, carry out flexible docking with cofactor pyridoxal 5＇-phosphate（PLP） and substrate cysteine and hereby detect its key residues. Through these procedures, the detail conformations of PLP-IscS（P-I） and cysteine-PLP-IscS（C-P-I） complexes were obtained. In P-I complex, the residues of Lys208, His106, Thr78, Ser205, His207, Asp182 and Gln185 have large interaction energies and/or hydrogen bonds fixation with PLP. In C-P-I complex, the amino group in cysteine is very near His106, Lys208 and PLP, the interaction energies for cysteine with them are very high. The above results are well consistent with those experimental facts of the homologues from other sources. Interestingly, the four residues of Glul05, Glu79, Ser203 and Hisl80 in P-I docking and the residue of Lys213 in C-P-I docking also have great interaction energies, which are fitly conservation in IscSs from all kinds of sources but have not been identified before. From these results, this gene can be confirmed at 3D level to encode the iron-sulfur cluster assembly protein IscS and subsequently play a sulfur traffic role. Furthermore, the substrate cysteine can be presumed to be effectively recruited into the active site. Finally, the above detected key residues can be conjectured to be directly responsible for the bind and/or catalysis of PLP and cysteine.展开更多
The gene iscS-2 from extremophile Acidithiobacillus ferrooxidans may play a crucial role in nitrogenase maturation. To investigate the protein encoded by this gene, a reliable integral three-dimensional molecular stru...The gene iscS-2 from extremophile Acidithiobacillus ferrooxidans may play a crucial role in nitrogenase maturation. To investigate the protein encoded by this gene, a reliable integral three-dimensional molecular structure was built. The obtained structure was further used to search binding sites, carry out the flexible docking with cofactor pyridoxal 5′-phosphate(PLP) and substrate cysteine, and identify its key residues. The docking results of PLP reveal that the residues of Lys203, His100, Thr73, Ser200, His202, Asp177 and Gln180 have large interaction energies and/or hydrogen bonds fixation with PLP. The docking results of cysteine show that the amino group in cysteine is very near His100, Lys203 and PLP, and the interaction energies for cysteine with them are very big. These identified residues are in line with the experimental facts of NifS from other sources. Moreover, the four residues of Asn152, Val179, Ala102 and Met148 in the PLP docking and the two residues of Lys208 and Ala102 in the cysteine docking also have large interaction energies, which are fitly conserved in NifS from all kinds of sources but have not been identified before. According to these results, this gene encodes NifS protein, and the substrate cysteine can be effectively recruited into the active site. Furthermore, all of the above detected key residues are directly responsible for the binding and/or catalysis of PLP and cysteine.展开更多
Objectives： Alliinase is a pyridoxal-5＇-phosphate （PLP）-dependent enzyme responsible for the production of diallyl thiosulfinate （allicin）, the biologically active component of garlic, from alliin. The use of al...Objectives： Alliinase is a pyridoxal-5＇-phosphate （PLP）-dependent enzyme responsible for the production of diallyl thiosulfinate （allicin）, the biologically active component of garlic, from alliin. The use of allicin for treatment of various diseases has been proposed but it is very unstable in the blood stream. This difficulty can be overcome by administration of alliin, together with a conjugate of alliinase directed towards the target cells. This, in turn requires a stable and active form of the enzyme. In this study we evaluate the stability of alliinase itself, in the presence mad absence of osmolytes, as well as that of its catalytically active complex with a mannose-specific lectin, ASAI （Allium sativum agglutinin I）, also presents in garlic. Methods： Alliinase, mad ASAI were both purified from garlic cloves. Thermal stability of alliinase itself, mad of its complexes with PLP and ASAI, in the presence mad absence of osmolytes, was analyzed by monitoring enzymic activity, and using DSC （differential scanning calorimetry）. Key findings： PLP exerts only a minor influence on alliinase structure and stablity. But both osmolytes and ASAI stabilize the enzyme considerably. Conclusions： The principle finding is that ASAI greatly stabilizes alliinase. Thus, the lectin-enzyme complex, which can be lyophilized and stored until used, provides an effective formulation of alliinase for generation of allicin from alliin in vivo.展开更多
基金This work was supported by the Foundation of Talented Person Development of Anhui Province in 2004.
文摘Pyridoxal kinase （PLK） （EC 2.7.1.35） catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5＇-phosphate （PLP）, an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method （GenBank accession number： DQ452397）. The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 k.Da. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.
文摘Colorectal cancer remains the third most common cancer in both women and men worldwide.Identifying modifiable dietary factors is crucial in developing primary prevention strategies.Vitamin B6 is involved in more than 100 coenzyme reactions,and may influence colorectal cancer risk in multiple ways including through its role in one-carbon metabolism related DNA synthesis and methylation and by reducing inflammation,cell proliferation,and oxidative stress.Observational studies of dietary or dietary plus supplementary intake of vitamin B6 and colorectal cancer risk have been inconsistent with most studies reporting nonsignificant positive or inverse associations.However,published studies of plasma pyridoxal 5'-phosphate(the active form of vitamin B6) levels consistently support an approximately 30%-50% reduction in risk of colorectal cancer comparing high with low concentrations.The reasons for the discrepancy in the results between dietary-based and plasma-based studies remain unresolved.Other unresolved questions include the effects of vitamin B6 intake in early life(i.e.,childhood or adolescence) and of suboptimal vitamin B6 status on colorectal cancer risk,whether the associations with vitamin B6 differ across molecular subtypes of colorectal cancer,and whether the vitamin B6-colorectal cancer association is modified by genetic variants of one-carbon metabolism.
基金Project(2004CB619201) supported by the National Basic Research Program of China Project(50321402) supported by the National Natural Science Foundation of China
文摘The gene iscS-3 from ,4cidithiobacillus ferrooxidans may play a central role in the delivery of sulfur to a variety of metabolic pathways in this organism. For insight into the sulfur metabolic mechanism of the bacteria, an integral three-dimensional （3D） molecular structure of the protein encoded by this gene was built by homology modeling techniques, refined by molecular dynamics simulations, assessed by PROFILE-3D and PROSTAT programs and further used to search bind sites, carry out flexible docking with cofactor pyridoxal 5＇-phosphate（PLP） and substrate cysteine and hereby detect its key residues. Through these procedures, the detail conformations of PLP-IscS（P-I） and cysteine-PLP-IscS（C-P-I） complexes were obtained. In P-I complex, the residues of Lys208, His106, Thr78, Ser205, His207, Asp182 and Gln185 have large interaction energies and/or hydrogen bonds fixation with PLP. In C-P-I complex, the amino group in cysteine is very near His106, Lys208 and PLP, the interaction energies for cysteine with them are very high. The above results are well consistent with those experimental facts of the homologues from other sources. Interestingly, the four residues of Glul05, Glu79, Ser203 and Hisl80 in P-I docking and the residue of Lys213 in C-P-I docking also have great interaction energies, which are fitly conservation in IscSs from all kinds of sources but have not been identified before. From these results, this gene can be confirmed at 3D level to encode the iron-sulfur cluster assembly protein IscS and subsequently play a sulfur traffic role. Furthermore, the substrate cysteine can be presumed to be effectively recruited into the active site. Finally, the above detected key residues can be conjectured to be directly responsible for the bind and/or catalysis of PLP and cysteine.
基金Project(2004CB619201) supported by the National Basic Research Program of ChinaProject(50321402) supported by the National Natural Science Foundation of China
文摘The gene iscS-2 from extremophile Acidithiobacillus ferrooxidans may play a crucial role in nitrogenase maturation. To investigate the protein encoded by this gene, a reliable integral three-dimensional molecular structure was built. The obtained structure was further used to search binding sites, carry out the flexible docking with cofactor pyridoxal 5′-phosphate(PLP) and substrate cysteine, and identify its key residues. The docking results of PLP reveal that the residues of Lys203, His100, Thr73, Ser200, His202, Asp177 and Gln180 have large interaction energies and/or hydrogen bonds fixation with PLP. The docking results of cysteine show that the amino group in cysteine is very near His100, Lys203 and PLP, and the interaction energies for cysteine with them are very big. These identified residues are in line with the experimental facts of NifS from other sources. Moreover, the four residues of Asn152, Val179, Ala102 and Met148 in the PLP docking and the two residues of Lys208 and Ala102 in the cysteine docking also have large interaction energies, which are fitly conserved in NifS from all kinds of sources but have not been identified before. According to these results, this gene encodes NifS protein, and the substrate cysteine can be effectively recruited into the active site. Furthermore, all of the above detected key residues are directly responsible for the binding and/or catalysis of PLP and cysteine.
文摘Objectives： Alliinase is a pyridoxal-5＇-phosphate （PLP）-dependent enzyme responsible for the production of diallyl thiosulfinate （allicin）, the biologically active component of garlic, from alliin. The use of allicin for treatment of various diseases has been proposed but it is very unstable in the blood stream. This difficulty can be overcome by administration of alliin, together with a conjugate of alliinase directed towards the target cells. This, in turn requires a stable and active form of the enzyme. In this study we evaluate the stability of alliinase itself, in the presence mad absence of osmolytes, as well as that of its catalytically active complex with a mannose-specific lectin, ASAI （Allium sativum agglutinin I）, also presents in garlic. Methods： Alliinase, mad ASAI were both purified from garlic cloves. Thermal stability of alliinase itself, mad of its complexes with PLP and ASAI, in the presence mad absence of osmolytes, was analyzed by monitoring enzymic activity, and using DSC （differential scanning calorimetry）. Key findings： PLP exerts only a minor influence on alliinase structure and stablity. But both osmolytes and ASAI stabilize the enzyme considerably. Conclusions： The principle finding is that ASAI greatly stabilizes alliinase. Thus, the lectin-enzyme complex, which can be lyophilized and stored until used, provides an effective formulation of alliinase for generation of allicin from alliin in vivo.
