title: 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.
authors.affiliation: Department of Chemistry, Macquarie University, Sydney NSW 2109, Australia.
authors.forename: Blair S
authors.initials: BS
authors.lastname: Nield
authors.forename: Robert D
authors.initials: RD
authors.lastname: Willows
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Torda
authors.forename: Michael R
authors.initials: MR
authors.lastname: Gillings
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Holmes
authors.forename: K M Helena
authors.initials: KM
authors.lastname: Nevalainen
authors.forename: H W
authors.initials: HW
authors.lastname: Stokes
authors.forename: Bridget C
authors.initials: BC
authors.lastname: Mabbutt
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004777:Environment; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D011994:Recombinant Proteins; D008780:Methyltransferases; D010770:Phosphotransferases