Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

Abstract

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:NM-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 13-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 1-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, lucD439, 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. The enzyme was stringently specific with regard to its substrate and cofactor, L-lysine and flavin adenine dinucleotide, respectively. NADPH appeared to be the preferred electron donor used by IucD439 in the N hydroxylation process.

Heterologous Expression, Purification, and Characterization of an l -Ornithine N 5 -Hydroxylase Involved in Pyoverdine Siderophore Biosynthesis in Pseudomonas aeruginosa

“…IucD was able to utilize NADH as a cofactor, although the activity of the enzyme was twofold lower than the corresponding activity with NADPH as a cofactor (22). PvdA, on the other hand, was not able to use NADH in place of NADPH as a cofactor.…”

Cloning, analysis, and overexpression of the gene encoding isobutylamine N-hydroxylase from the valanimycin producer, Streptomyces viridifaciens

“…A key step in these pathways involves the N 5 -hydroxylation of L-ornithine in the case of pyoverdin (17,34) and the N 6 -hydroxylation of L-lysine in the case of aerobactin (22,29,30). These hydroxylations are catalyzed by flavin monooxygenases.…”

A genetic locus required for iron acquisition in Mycobacterium tuberculosis

“…The mbtG gene product from the mbt cluster has been anticipated to be a lysine N6-hydroxylase (9,15). Previous studies with N6-hydroxylase from aerobactin biosynthetic cluster had suggested that these proteins belong to the family of flavoprotein monooxygenases and consume molecular oxygen during catalysis (31,32). The enzymatic reactions with MbtG protein were carried out with N6-lauroyl Z-Lys-OMe (II) in the presence of NADPH and FAD, in air saturated buffers (Fig.…”