Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family.
The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.
Robinson
Andrew
A
Department of Chemistry, Macquarie University, NSW 2109, Australia.
Wu
Peter S-C
PS
Harrop
Stephen J
SJ
Schaeffer
Patrick M
PM
Dosztányi
Zsuzsanna
Z
Gillings
Michael R
MR
Holmes
Andrew J
AJ
Nevalainen
K M Helena
KM
Stokes
H W
HW
Otting
Gottfried
G
Dixon
Nicholas E
NE
Curmi
Paul M G
PM
Mabbutt
Bridget C
BC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004251:DNA Transposable Elements; D004851:Epoxide Hydrolases / Q000737:chemistry; D032023:Integrons / Q000502:physiology*; D017136:Ion Transport; D007535:Isomerases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D012987:Soil; D004851:Epoxide Hydrolases; C115926:limonene-1,2-epoxide hydrolase; D007535:Isomerases; C405498:nogalonic acid methyl ester cyclase