Exploring nitrilase sequence space for enantioselective catalysis.
Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10(6) to 10(10) members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.
Robertson
Dan E
DE
Diversa Corporation, San Diego, California 92121, USA. drobertson@diversa.com
Chaplin
Jennifer A
JA
DeSantis
Grace
G
Podar
Mircea
M
Madden
Mark
M
Chi
Ellen
E
Richardson
Toby
T
Milan
Aileen
A
Miller
Mark
M
Weiner
David P
DP
Wong
Kelvin
K
McQuaid
Jeff
J
Farwell
Bob
B
Preston
Lori A
LA
Tan
Xuqiu
X
Snead
Marjory A
MA
Keller
Martin
M
Mathur
Eric
E
Kretz
Patricia L
PL
Burk
Mark J
MJ
Short
Jay M
JM
D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D004783:Environmental Microbiology; D015723:Gene Library; D008969:Molecular Sequence Data; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D013237:Stereoisomerism; D013379:Substrate Specificity
D009570:Nitriles; D000619:Aminohydrolases; C021867:nitrilase