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Protein Science (2004), 13:1651-1659. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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New enzymes from environmental cassette arrays: Functional attributes of a phosphotransferase and an RNA-methyltransferase

Blair S. Nield1, Robert D. Willows2, Andrew E. Torda3, Michael R. Gillings2, Andrew J. Holmes2, K.M. Helena Nevalainen2, H.W. Stokes2 and Bridget C. Mabbutt1

1 Departments of Chemistry and
2 Biological Sciences, Macquarie University, Sydney NSW 2109, Australia
3 Centre for Bioinformatics, University of Hamburg, D-20146 Hamburg, Germany

(RECEIVED January 18, 2004; FINAL REVISION January 18, 2004; ACCEPTED February 25, 2004)



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 Mg2+-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.

Keywords: protein prospecting; gene cassette; APH; SpoU family; integron

Abbreviations: 59-be, 59-base element DNA recombination site • AdoMet, S-adenosyl-L-methionine • E-value, expectation value defined by homology search program • eAPH, aminoglycoside phosphotransferase from an environmental microbial source • eTrm, RNA 2'-O-ribose methyl-transferase from an environmental microbial source • GST, glutathione-S-transferase • MPH, macrolide 2-phosphotransferase • ORF, open reading frame • PCR, polymerase chain reaction • PDB, Protein Data Bank • RlmB, methyltransferase from Escherichia coli • RrmA, putative methyltranferase from Thermus thermophilus • RNase A, ribonuclease A • TCA, trichloroacetic acid • tRNA, transfer RNA • YibK, methyltransferase from Haemophilus influenzae

Reprint requests to: Bridget C. Mabbutt, Department of Chemistry, Macquarie University, Sydney NSW 2109, Australia; e-mail: bridget.mabbutt{at}mq.edu.au; fax: 61-2-9850-8313.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04638704.


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