Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus

Giddings, Lesley-Ann; Lountos, George T.; Kim, Kang Woo; Brockley, Matthew; Needle, Danielle; Cherry, Scott; Tropea, Joseph E.; Waugh, David S.

doi:10.1371/journal.pone.0248385

Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus

Journal Article · Tue Mar 30 00:00:00 EDT 2021 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0248385· OSTI ID:1775469

^[1]; ^[2]; Kim, Kang Woo ^[3]; Brockley, Matthew ^[3]; Needle, Danielle ^[4]; Cherry, Scott ^[4]; Tropea, Joseph E. ^[4]; Waugh, David S. ^[4]

Smith College, Northampton, MA (United States); Middlebury College, VT (United States)
Frederick National Lab. for Cancer Research, Frederick, MD (United States)
Middlebury College, VT (United States)
National Cancer Institute, Frederick, MD (United States)

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP⁺ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent k_cat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s^-1 and 0.24 ± 0.01 s^-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent k_cat for NADPH (0.034 ± 0.008 s^-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH K_M was 33 ± 24 μM. Under saturating FAD and NADPH concentrations, the apparent k_cat and K_M for cadaverine in Csaky assays were 0.048 ± 0.004 s^-1 and 19 ± 9 μM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH)

Grant/Contract Number:: W-31109-ENG-38

OSTI ID:: 1775469

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 3 Vol. 16; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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