ABSTRACT
Escherichia coli DnaJ, possessing both chaperone and thiol-disulfide oxidoreductase activities, is a homodimeric Hsp40 protein. Each subunit contains four copies of a sequence of -CXXCXGXG-, which coordinate with two Zn(II) ions to form an unusual topology of two C4-type zinc fingers, C144DVC147Zn(II)C197NKC200 (Zn1) and C161PTC164Zn(II)C183PHC186 (Zn2). Studies on five DnaJ mutants with Cys in Zn2 replaced by His or Ser (C183H, C186H, C161H/C183H, C164H/183H, and C161S/C164S) reveal that substitutions of one or two Cys residues by His or Ser have little effect on the general conformation and association property of the molecule. Replacement of two Cys residues by His does not interfere with the zinc coordination. However, replacement of two Cys by Ser results in a significant decrease in the proportion of coordinated Zn(II), although the unique zinc finger topology is retained. The mutants of C183H, C186H, and C161S/C164S display full disulfide reductase activity of wild-type DnaJ, while C161H/C183H and C164H/183H exhibit severe defect in the activity. All of the mutations do not substantially affect the chaperone activity. The results indicate that the motif of -CXXC- is critical to form an active site and indispensable to the thiol-disulfide oxidoreductase activity of DnaJ. Each -CXXC- motif in Zn2 but not in Zn1 functions as an active site.
Subject(s)
Cysteine/chemistry , Escherichia coli Proteins/chemistry , Heat-Shock Proteins/chemistry , Protein Disulfide Reductase (Glutathione)/chemistry , Zinc Fingers , Amino Acid Motifs/genetics , Amino Acid Sequence , Amino Acid Substitution/genetics , Binding Sites/genetics , Catalysis , Cysteine/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , HSP40 Heat-Shock Proteins , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Histidine/genetics , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation , Protein Disulfide Reductase (Glutathione)/genetics , Protein Disulfide Reductase (Glutathione)/metabolism , Protein Structure, Tertiary/genetics , Serine/genetics , Zinc/chemistry , Zinc Fingers/geneticsABSTRACT
Thioredoxin, DsbA, the N-terminal active-site domain a and the non-active-site domain b of protein-disulfide isomerase are all monomeric with a thioredoxin fold, and each exhibits low or no isomerase and chaperone activity. We have linked the N terminus of the above four monomers, individually, to the C terminus of the N-terminal domain of DsbC via the flexible linker helix of the latter to produce four domain hybrids, DsbCn-Trx, DsbCn-DsbA, DsbCn-PDIa, and DsbCn-PDIb. These four hybrid proteins form homodimers, and except for DsbCn-PDIb they exhibit new or greatly elevated isomerase as well as chaperone activity. Three-dimensional structure prediction indicates that all the four domain hybrids adopt DsbC-like V-shaped structure with a broad uncharged cleft between the two arms for binding of non-native protein folding intermediates. The results provide strong evidence that dimerization creates chaperone and isomerase activity for monomeric thiol-protein oxidases or reductases, and suggesting a pathway for proteins to acquire new functions and/or higher biological efficiency during evolution.
Subject(s)
Molecular Chaperones/chemistry , Binding Sites , Circular Dichroism , Dimerization , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Guanidine/chemistry , Models, Molecular , Plasmids/metabolism , Protein Binding , Protein Conformation , Protein Disulfide-Isomerases/chemistry , Protein Folding , Protein Structure, Tertiary , Thioredoxins/chemistry , Time FactorsABSTRACT
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.
Subject(s)
Amidohydrolases/metabolism , Arthrobacter/genetics , Escherichia coli/metabolism , Amidohydrolases/genetics , Arthrobacter/enzymology , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Genetic Vectors/genetics , Hydantoins/metabolism , Models, Genetic , Phenylalanine/metabolism , Plasmids/genetics , Polymerase Chain ReactionABSTRACT
Hydantoinase can be widely used in enzymic production of various amino acids. In order to obtain the hydantoinase genes in Arthrobacter BT801, its chromatosomal DNA is isolated and partialy digested with Sau3A I to collect fragments of about 30kb. Then, this fragment is inserted into the Hpa I and Pst I site of cosmid pKC505. The genomic library was thus constructed by packing in vitro with lambda phage package protein and transfecting E. coli DH5alpha. A positive transformant was selected from the library using thin layer chromatography and other methods. A DNA fragment containing complete hydantoinase genes was sequenced by sub-cloning into pUC18. The gene can express active protein under control of its own promoter and T5 promoter in E. coli. The isolation of the gene established foundition for research and application of the hydantoinase.
