The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family
Abstract
Iron is essential for growth and in low iron environments such as serum many bacteria and fungi secrete ferric iron-chelating molecules called siderophores. All fungi produce hydroxamate siderophores with the exception of Mucorales fungi, which secrete rhizoferrin, a polycarboxylate siderophore. Here we investigated the biosynthesis of rhizoferrin by the opportunistic human pathogen, Rhizopus delemar. We searched the genome of R. delemar 99–880 for a homologue of the bacterial NRPS-independent siderophore (NIS) protein, SfnaD that is involved in biosynthesis of staphyloferrin A in Staphylococcus aureus. A protein was identified in R. delemar with 22% identity and 37% similarity with SfnaD, containing an N-terminal IucA/IucC family domain, and a C-terminal conserved ferric iron reductase FhuF-like transporter domain. Expression of the putative fungal rhizoferrin synthetase (rfs) gene was repressed by iron. The rfs gene was cloned and expressed in E.coli and siderophore biosynthesis from citrate and diaminobutane was confirmed using high resolution LC–MS. Substrate specificity was investigated showing that Rfs produced AMP when oxaloacetic acid, tricarballylic acid, ornithine, hydroxylamine, diaminopentane and diaminopropane were employed as substrates. Based on the production of AMP and the presence of a mono-substituted rhizoferrin, we suggest that Rfs is a member of the superfamily of adenylating enzymes. We used site-directed mutagenesis to mutate selected conserved residues predicted to be in the Rfs active site. These studies revealed that H484 is essential for Rfs activity and L544 may play a role in amine recognition by the enzyme. This study on Rfs is the first characterization of a fungal NIS enzyme. Future work will determine if rhizoferrin biosynthesis is required for virulence in Mucorales fungi.
Citation
Carroll , C S , Grieve , C L , Murugathasan , I , Bennet , A J , Melo Czekster , C , Lui , H , Naismith , J & Moore , M M 2017 , ' The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family ' , International Journal of Biochemistry and Cell Biology , vol. 89 , pp. 136-146 . https://doi.org/10.1016/j.biocel.2017.06.005
Publication
International Journal of Biochemistry and Cell Biology
Status
Peer reviewed
ISSN
1357-2725Type
Journal article
Rights
© 2017 Elsevier Ltd. All rights reserved. This work has been made available online in accordance with the publisher’s policies. This is the author created, accepted version manuscript following peer review and may differ slightly from the final published version. The final published version of this work is available at https://doi.org/10.1016/j.biocel.2017.06.005
Description
This work was supported by the Natural Sciences and Engineering Research Council of Canada award to MM (grant number 611181). C. Carroll thanks Simon Fraser University for a travel and research award.Collections
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