New enzymes from environmental cassette arrays: functional attributes of a phosphotransferase and an RNA-methyltransferase

Abstract

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7") from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg(2+)-binding residues. Unlike related APH(4) or APH(7") enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.

Figure 1.

Structure-based alignments for environmentally derived APH (eAPH) and RNA 2′-O-methyltransferase (eTrm) orthologs. (A) Alignment for eAPH includes homologous APH sequences spanning the evolutionary subfamilies identified by Wright and Thompson (1999): APH(7″), S. hygroscopicus hygromycin B phosphotransferase (SwissProt P09979); MPH, E. coli macrolide 2′-phosphotransferase I (SwissProt BAA03776); and APH(3′), E. faecalis aminoglycoside phosphotransferase (3′)-IIIa (SwissProt P00554). Helices and arrows indicate secondary structure features of APH(3′)-IIIa (Hon et al. 1997) in PDB file 1J7I. Asterisks indicate key conserved residues interacting with ATP and Mg²⁺ in the active site. (B) Alignment of eTrm sequence with known structures of H. influenzae YibK (PDB 1J85A), T. thermophilus RrmA (PDB 1IPAA), and E. coli RlmB (PDB 1GZ0). Residues occurring within α-helices and β-strands are outlined in black and in gray, respectively. Sequence motifs I–III are those used by Gustafsson et al. (1996) to define the 2′-O-methyltransferases.

Figure 1.

Structure-based alignments for environmentally derived APH (eAPH) and RNA 2′-O-methyltransferase (eTrm) orthologs. (A) Alignment for eAPH includes homologous APH sequences spanning the evolutionary subfamilies identified by Wright and Thompson (1999): APH(7″), S. hygroscopicus hygromycin B phosphotransferase (SwissProt P09979); MPH, E. coli macrolide 2′-phosphotransferase I (SwissProt BAA03776); and APH(3′), E. faecalis aminoglycoside phosphotransferase (3′)-IIIa (SwissProt P00554). Helices and arrows indicate secondary structure features of APH(3′)-IIIa (Hon et al. 1997) in PDB file 1J7I. Asterisks indicate key conserved residues interacting with ATP and Mg²⁺ in the active site. (B) Alignment of eTrm sequence with known structures of H. influenzae YibK (PDB 1J85A), T. thermophilus RrmA (PDB 1IPAA), and E. coli RlmB (PDB 1GZ0). Residues occurring within α-helices and β-strands are outlined in black and in gray, respectively. Sequence motifs I–III are those used by Gustafsson et al. (1996) to define the 2′-O-methyltransferases.

Figure 2.

Production and phosphotransferase activity of recombinant GST-eAPH (GST-aminoglycoside phosphotransferase ortholog from orf346_Pu5). (A) SDS PAGE-gel (visualized with silver stain) shows: Lanes M, 10-kD MW ladder; 1, pre-induced BL21 E. coli cells; 2, IPTG-induced cells; 3, soluble cell fraction; 4, inclusion bodies; 5, desalted GST-eAPH (98% purity) following refolding from urea-solubilized inclusion bodies. (B) ATPase activity assay of purified GST-eAPH. Samples contained [8-¹⁴C]-ATP alone, or [8-¹⁴C]-ATP in the presence of GST-eAPH with and without aminoglycoside substrate. Reactions were incubated at room temperature, then the nucleotides were separated by TLC and visualized by autoradiography. Percentage degradation of ATP to ADP and AMP was determined by densitometry. Error bars indicate standard deviation.

Figure 3.

Production and methyltransferase activity of recombinant His₆-eTrm (tRNA methyltransferase ortholog from orf208_Bal31). (A) SDS PAGE-gel (visualized with Coomassie dye) shows: Lanes M, 10-kD MW ladder; 1, pre-induced BL21(De3)/pLysS E. coli cells; 2, IPTG-induced cells; 3, soluble cell fraction; 4, insoluble fraction; 5, His₆-eTrm following elution from Ni-NTA agarose. (B) Incorporation of [³H-CH₃] into tRNA in the presence of purified His₆-eTrm at room temperature. Incubation of His₆-eTrm (4μM) and [³H]-AdoMet (40 μM) was carried out with varying amounts of tRNA (E. coli strain W) in 100 mM Tris buffer (pH 7.5) (containing 40 mM NH₄Cl, 2 mM MgCl₂, and 5 mM DTT). After 50 min, reactions were stopped and filtered through nylon membrane filters. Methylation activity was determined by scintillation counting and is given as nmoles of [³H-CH₃] groups incorporated/h/mg protein. To account for nonspecific interactions, the background value obtained for enzyme without tRNA was subtracted from all test assays.