Functional and kinetic analysis of the phosphotransferase CapP conferring selective self-resistance to capuramycin antibiotics

Abstract

Capuramycin-related compounds, including A-500359s and A-503083s, are nucleoside antibiotics that inhibit the enzyme bacterial translocase I involved in peptidoglycan cell wall biosynthesis. Within the biosynthetic gene cluster for the A-500359s exists a gene encoding a putative aminoglycoside 3-phosphotransferase that was previously demonstrated to be highly expressed during the production of A-500359s and confers selective resistance to capuramycins when expressed in heterologous hosts. A similar gene (capP) was identified within the biosynthetic gene cluster for the A-503083s, and CapP is now shown to similarly confer selective resistance to capuramycins. Recombinant CapP was produced and purified from Escherichia coli, and the function of CapP is established as an ATP-dependent capuramycin phosphotransferase that regio-specifically transfers the gamma-phosphate to the 3''-hydroxyl of the unsaturated hexuronic acid moiety of A-503083 B. Kinetic analysis with the three major A-503083 congeners suggests that CapP preferentially phosphorylates A-503083s containing an aminocaprolactam moiety attached to the hexuronic acid, and bi-substrate kinetic analysis was consistent with CapP employing a sequential kinetic mechanism similar to most known aminoglycoside 3-phosphotransferases. The purified CapP product lost its antibiotic activity against Mycobacterium smegmatis, and this loss in bioactivity is primarily due to a 272-fold increase in the IC(50) in the bacterial translocase I-catalyzed reaction. The results establish CapP-mediated phosphorylation as a mechanism of resistance to capuramycins and now set the stage to explore this strategy of resistance as a potential mechanism inherent to pathogens and provide the impetus for preparing second generation analogues as a preemptive strike to such resistance strategies.

FIGURE 1.

Structures of representative capuramycins that were discovered using a screen to isolate specific inhibitors of MraY.

FIGURE 2.

Heterologous expression of capP conferring resistance to A-500359 B. A, S. albus J1074 with pWHM3-Ep (control) or pWHM3-Ep-capP grown on ISP2 media containing different concentrations of antibiotic. 1/100 and 1/1000 indicate dilution ratios of cultured mycelium spotted on the agar media. B, E. coli ΔtolC with pUC19 (control) or pUC19-capP grown on LB media containing different concentrations of antibiotic and 1 mm isopropyl 1-thio-β-d-galactopyranoside for induction of expression.

FIGURE 3.

In vitro characterization of CapP. A, SDS-PAGE of purified CapP (expected molecular mass of 37.8 kDa). Lane 1, molecular mass markers; lane 2, purified His₆-CapP. B–D, HPLC analysis of the activity of CapP showing the time-dependent formation of a new peak with a mass consistent with the mono-phosphorylated product and the simultaneous loss of the substrate peak.

FIGURE 4.

Biological activity of 3″-phospho-A-503083 B. A, antimicrobial activity of A-503083 B and the CapP product using the test strain M. smegmatis SANK 70186. The control consists of solvent only (dimethyl sulfoxide/methanol, 7:3). B, plot of the relative activity of MraY with variable A-503083 B or CapP product.

FIGURE 5.

Kinetic analysis of CapP-catalyzed reaction. A, single-substrate kinetic analysis using near saturating ATP and variable A-503083 A. B, single-substrate kinetic analysis using near saturating ATP and variable A-503083 B. C, single-substrate kinetic analysis using near saturating A-503083 B and variable ATP. D, bi-substrate kinetic analysis using variable ATP and multiple fixed concentration of A-503083 B at 1 μm (●), 10 μm (▲), 50 μm (▼), and 200 mm μm (○).