Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution.
Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences.
Yang
H
H
Research School of Chemistry, Australian National University, GPO Box 414, Canberra, ACT 2601, Australia.
Carr
P D
PD
McLoughlin
S Yu
SY
Liu
J W
JW
Horne
I
I
Qiu
X
X
Jeffries
C M J
CM
Russell
R J
RJ
Oakeshott
J G
JG
Ollis
D L
DL
D000595:Amino Acid Sequence; D001665:Binding Sites; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D019020:Directed Molecular Evolution; D019143:Evolution, Molecular; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism*; D010626:Phenylethyl Alcohol / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D003035:Cobalt; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases; D044345:Phosphoric Triester Hydrolases; D010626:Phenylethyl Alcohol