Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster.
The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)
Thariath
A
A
Guelph-Waterloo Centre for Graduate Work in Chemistry, University of Waterloo, Ontario, Canada.
Socha
D
D
Valvano
M A
MA
Viswanatha
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006877:Hydroxamic Acids / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009994:Osmolar Concentration; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D013329:Structure-Activity Relationship
D006877:Hydroxamic Acids; D011994:Recombinant Proteins; C031819:aerobactin; D006899:Mixed Function Oxygenases; C047196:lysine N-epsilon hydroxylase