The Final Step of Hygromycin A Biosynthesis, Oxidation of C-5″-Dihydrohygromycin A, Is Linked to a Putative Proton Gradient-Dependent Efflux

Abstract

Hygromycin A (HA) is an aminocyclitol antibiotic produced and excreted by Streptomyces hygroscopicus. Deletion of hyg26 from the hygromycin A biosynthetic gene cluster has previously been shown to result in a mutant that produces 5؆-dihydrohygromycin A (DHHA). We report herein on the purification and characterization of Hyg26 expressed in Escherichia coli. The enzyme catalyzes an NAD(H)-dependent reversible interconversion of HA and DHHA, supporting the role of the reduced HA as the penultimate biosynthetic pathway intermediate and not a shunt product. The equilibrium for the Hyg26-catalyzed reaction heavily favors the DHHA intermediate. The high-titer production of the HA product by S. hygroscopicus must be dependent upon a subsequent energetically favorable enzyme-catalyzed process, such as the selective and efficient export of HA. hyg19 encodes a putative proton gradient-dependent transporter, and a mutant lacking this gene was observed to produce less HA and to produce the DHHA intermediate. The DHHA produced by either the ⌬hyg19 or the ⌬hyg26 mutant had slightly reduced activity against E. coli and reduced protein synthesis-inhibitory activity in vitro. The data indicate that Hyg26 and Hyg19 have evolved to produce and export the final potent HA product in a coordinated fashion.

Distinct tRNA Accommodation Intermediates Observed on the Ribosome with the Antibiotics Hygromycin A and A201A

“…3B). Consistent with previous studies (Dhote et al, 2009; Guerrero and Modolell, 1980; Palaniappan et al, 2009), HygA displayed strong inhibitory properties, inhibiting GFP production with a half inhibitory concentration (IC 50 ) of 0.2 μM (Fig. 3B).…”

“…The strain of S. hygroscopicus that produces HygA is thought to obtain self-resistance to HygA using at least two mechanisms: (i) efflux of the drug by the putative proton gradient-dependent efflux pumps Hyg19 and Hyg28 (Dhote et al, 2009); (ii) inactivation of HygA through the action of the phosphotransferase Hyg21 (Dhote et al, 2008). The latter catalyzes the transfer of the γ-phosphoryl group from ATP to the ribose 2″-OH of subunit A of HygA (Fig.…”

“…The total synthesis of HygA (Chida et al, 1989; Donohoe et al, 2009) and, more recently, A201A (Nie et al, 2014) as well as the ability to generate biosynthetic precursors with biological activity (Dhote et al, 2009; Habib el et al, 2003; Palaniappan et al, 2006; Palaniappan et al, 2009), has further opened the way to developing successive generations of these antibiotics with improved antimicrobial properties. However, to fully understand the structure-activity relationships of HygA, A201A and analogs thereof, insights into the molecular modes by which these antibiotics interact with the ribosome and inhibit translation are required.…”

Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A

“…If the furanose moiety is maintained, the tight packing of the 2″ and 3″ hydroxyl groups with the walls of the pocket is in agreement with previous work showing that the 3″ OH is essential ( 55 ) and the 2″ OH is phosphorylated in the HygA producing organism in one of several possible mechanisms of self-resistance ( 14 ). Furthermore the acetyl group of the furanose moiety is amendable to modification (as seen with compounds 5″-dihydrohygromycin A) ( 57 ), which is in agreement with the fact that this group extends into the lumen of the ribosomal tunnel. In the cinnamic acid moiety the strict steric requirements imposed on active substituents ( 8 , 58 , 59 ) is supported by the structure which indicates this moiety is tightly surrounded on one face by the 23S rRNA and on the other by the P-site bound tRNA (as inferred by aligning structures harboring P-site substrates to that of the HygA structure).…”

Characterization of the biosynthetic gene cluster (ata) for the A201A aminonucleoside antibiotic from Saccharothrix mutabilis subsp. capreolus

“…5 The structural basis for both Hygromycin A and A201A antibiotics binding and inhibition to ribosomes have been recently showed by Polikanov et al 6 The similarities of A201A structure with puromycin and hygromycin A antibiotics strongly suggest that certain enzymes, and hence the corresponding genes in the A201A biosynthetic pathway, may be related to their counterparts in the puromycin and hygromycin A biosynthetic pathways. [7][8][9][10][11] Homologs of the ata open reading frames (ORFs) have indeed been found with for at least 14 ORFs of the hygromycin A biosynthetic cluster in S. hygroscopicus 9 and as we showed previously a set of five consecutive genes involved in the biosynthesis of the aminonucleoside moiety of A201A 12 and their deduced products (AtaP3, AtaP4, AtaP5, AtaP7 and AtaP10) are similar to their counterparts from the pur cluster of S. alboniger (Pur3, Pur4, Pur5, Pur7 and Pur10, respectively), genes implicated in the biosynthesis of the aminonucleoside moiety of puromycin. Not surprisingly, ataP4, ataP5 and ataP10 can complement corresponding mutations in the pur cluster in S. alboniger.…”