Biochemical Characterization of New Microbioal Dehalogenases

1
Biochemical Characterization of New Microbioal
Dehalogenases
Chan W. Y.1,3, Guthrie J.2, Korniyenko Y.1,
Savchenko A.1,4, Pai E. F.1,4, Edwards E. A.5 and
Yakunin A. F.1,41. Banting and Best Department
of Medical Research, University of Toronto,
Toronto, Canada2. Clinical Genomics Centre,
University Health Network, Toronto, Canada3.
Department of Biochemistry, University of
Toronto, Toronto, Canada4. Ontario Centre for
Structural Proteomics, Ontario Cancer Institute,
Toronto, Canada5. Department of Chemical
Engineering and Applied Chemistry, University of
Toronto, Canada
Conserved motif alignment of haloalkane
dehalogenases
Abstract Synthetic organohalogens represent an
important class of xenobiotics that pose major
environmental threats due to their toxicity and
persistence. Genome sequencing has uncovered
over 3,000 proteins with sequence similarity to
dehalogenases, but fewer than 50 have been
characterized. Our objective is to discover new
microbial dehalogenases capable of degrading
important environmental xenobiotics, with greater
efficiency and wider substrate range than the
known enzymes. We selected 300 dehalogenase-like
genes from sequenced genomes of Pseudomonas
aeruginosa, Ps. putida, Rhodococcus sp.,
Rhodopseudomonas palustris, Sinorhizobium
meliloti, Streptomyces avermitilis and St.
coelicolor. They belong to two large classes of
dehalogenases haloalkane (Type I and II) and
(S)-2-haloacid dehalogenases. Sequence
alignments have highlighted several conserved
motifs such as the N-terminal beta strand
R-(A/I/M)-H-(H/Y)-(L/V)-DEG and the catalytic
motif QDWG in Type I haloalkane dehalogenases.
In Type II haloalkane dehalogenases, this beta
strand is less conserved and the catalytic motif
is better described by (H/Q)-D-(W/Y)-G. In
(S)-2-haloacid dehalogenases, the N-terminus,
which harbours the catalytic nucleophile,
contains many highly conserved regions (e.g.
FD-(A/L/M)-YGTL-(C/F/L/Y)-DV). These selected
proteins were purified and screened for
dehalogenase, phosphatase, esterase and
thioesterase activity against general substrates
(e.g. haloacetates, 1,2-dichloroethane, pNPP,
pNP-palmitate and palmitoyl-CoA) and preliminary
results will be presented.
Functional identification of purified proteins
- Haloacid dehalogenase screen with chloroacetate
PA0810
SM0968
Sequence analysis of existing haloalkane
dehalogenases reveal many highly conserved
motifs, two of which correspond to the catalytic
motif and structural motif.
L-Dex
- HAD-like phosphatase screen with pNPP
Conserved motif alignment of newly identified
haloacid dehalogenases
The 4 HADs all show a high degree of sequence
similarity with other members of the family.
However, it is interesting that the St.
avermitilis HAD, which has a unique residue at
several positions that would otherwise be highly
conserved, is very inefficient at dehalogenating
chloroacetate in the preliminary screens.
- Activity profile of purified proteins
General reaction mechanisms
- Haloalkane dehalogenase
Conclusion To date, 233 genes were cloned from
various organisms and 49 well-expressed soluble
proteins have been purified from a total of 65
purifications. Through general substrate
activity screening, we have identified 4 haloacid
dehalogenases, 1 esterase, 3 thioesterases and 10
phosphatases. Further characterization will be
performed to identify more dehalogenases.
- Haloacid dehalogenase
Funding 1. NSERC Strategic Project Grant 2.
Genome Canada II
In both dehalogenases, the reaction begins with
a SN2 attack by the catalytic nucleophile on the
halogenated carbon, followed by the turnover of
the ester enzyme-substrate intermediate by a
nucleophilic water.