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Antimicrobial Agents and Chemotherapy, October 2008, p. 3580-3588, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00157-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

An O-Phosphotransferase Catalyzes Phosphorylation of Hygromycin A in the Antibiotic-Producing Organism Streptomyces hygroscopicus{triangledown}

Vidya Dhote, Shuchi Gupta, and Kevin A. Reynolds*

Department of Chemistry, Portland State University, Portland, Oregon 97201

Received 4 February 2008/ Returned for modification 21 April 2008/ Accepted 20 June 2008

The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (Km = 30 ± 4 µM) at the C-2''' position of the fucofuranose ring in the presence of ATP (Km = 200 ± 20 µM) or GTP (Km = 350 ± 60 µM) with a kcat of 2.2 ± 0.1 min–1. The phosphorylated HA is inactive against HA-sensitive {Delta}tolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with kcat and Km values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5'''-dihydrohygromycin A and 5'''-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388.

* Corresponding author. Mailing address: Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207-0751. Phone: (503) 725-3886. Fax: (503) 725-9525. E-mail: reynoldsk{at}pdx.edu

{triangledown} Published ahead of print on 21 July 2008.

Antimicrobial Agents and Chemotherapy, October 2008, p. 3580-3588, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00157-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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