title: Exploring nitrilase sequence space for enantioselective catalysis.
abstract: 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.
authors.affiliation: Diversa Corporation, San Diego, California 92121, USA. drobertson@diversa.com
authors.forename: Dan E
authors.initials: DE
authors.lastname: Robertson
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Chaplin
authors.forename: Grace
authors.initials: G
authors.lastname: DeSantis
authors.forename: Mircea
authors.initials: M
authors.lastname: Podar
authors.forename: Mark
authors.initials: M
authors.lastname: Madden
authors.forename: Ellen
authors.initials: E
authors.lastname: Chi
authors.forename: Toby
authors.initials: T
authors.lastname: Richardson
authors.forename: Aileen
authors.initials: A
authors.lastname: Milan
authors.forename: Mark
authors.initials: M
authors.lastname: Miller
authors.forename: David P
authors.initials: DP
authors.lastname: Weiner
authors.forename: Kelvin
authors.initials: K
authors.lastname: Wong
authors.forename: Jeff
authors.initials: J
authors.lastname: McQuaid
authors.forename: Bob
authors.initials: B
authors.lastname: Farwell
authors.forename: Lori A
authors.initials: LA
authors.lastname: Preston
authors.forename: Xuqiu
authors.initials: X
authors.lastname: Tan
authors.forename: Marjory A
authors.initials: MA
authors.lastname: Snead
authors.forename: Martin
authors.initials: M
authors.lastname: Keller
authors.forename: Eric
authors.initials: E
authors.lastname: Mathur
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Kretz
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Burk
authors.forename: Jay M
authors.initials: JM
authors.lastname: Short
mesh_terms: 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
chemical_list: D009570:Nitriles; D000619:Aminohydrolases; C021867:nitrilase