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overexpression in E. coli BL21(DE3), enzyme activity and enantiose-
lectivity towards p-chlorothioanisole were determined (as listed in
Table S1).
P450RhF, was also reported to display a high degree of substrate
promiscuity.[24] Nevertheless, no highly regio- and enantioselec-
tive hydroxylation of alkanes by P450RhF has been reported so
far.
Cloning, expression, and purification: For the expression and pu-
rification of the P450s, E. coli BL21(DE3) cells harboring plasmid
pET28a-His-P450 (with a His6-tag at the N terminus) were used. The
primers used for each gene target encoding a P450 are listed in
Table S2. The resulting pET28-P450 plasmids were transformed into
E. coli BL21(DE3) cells. The cells were grown in Terrific Broth
medium [500 mL, containing kanamycin (50 mgmLÀ1)] at 378C to
an optical density of 0.8 at 600 nm. IPTG (0.2 mm) and d-amino-
levulinic acid (d-ALA, 0.5 mm) were then added to induce protein
expression, and the culture was incubated for another 24 h at
168C. The cells were harvested by centrifugation at 5000g for
20 min, washed twice with saline, resuspended in buffer A [sodium
phosphate buffer (pH 8.0, 50 mm) containing glycerol (10%, v/v),
NaCl (500 mm), imidazole (10 mm)] and lysed on ice with an ultra-
sonic oscillator (JY92-II, Scientz Biotech. Co., Ltd.). After centrifuga-
tion (8000g for 20 min), the cell lysate supernatant was filtered
and loaded onto a His trap Ni-NTA FF column (5 mL, GE Health-
care), pre-equilibrated with buffer A. Proteins were eluted with an
increasing gradient of imidazole (10 to 250 mm) in buffer A at
a flow rate of 1 mLminÀ1. Fraction purities were determined by
SDS-PAGE, and all fractions containing target protein were collect-
ed and dialyzed against sodium phosphate buffer (pH 8.0, 50 mm)
for desalting. The concentration of P450 protein was determined
by CO difference spectroscopy as described elsewhere.[10] Finally,
the sample was concentrated and stored at À808C in glycerol
(20%) until further use.
Enzymes with more promiscuous substrate selectivity are
more likely to prove useful as templates for directed protein
evolution than less promiscuous enzymes with greater activity,
as long as the desired activities are already present.[25] The po-
tential for diverse applications of the P450s has prompted ex-
tensive protein engineering research directed towards enhanc-
ing their activity or coupling efficiencies and improving their
selectivities. The best example of this is P450Bm3; numerous var-
iants for the conversion of a diverse range of substrates have
been produced by random mutagenesis or by rational design
based on the crystal structure of this enzyme.[26] Although no
crystal structures for the P450-PDOR-like fusion enzymes of the
CYP116B subfamily are yet available, the self-sufficient nature
of P450LaMO coupled with its newly established broad substrate
promiscuity highlight it as an ideal template for directed evolu-
tion. Our future work will therefore focus on the crystallization
and evolution of P450LaMO to provide a more robust biocatalyst
with higher activity and selectivity.
Conclusion
We have presented the discovery of a new self-sufficient P450
by genome mining with the purpose of finding a monooxyge-
nase with new substrate specificities. This strategy yielded
a versatile monooxygenase, P450LaMO, which exhibits an unusu-
ally broad substrate range for different oxidation reactions in-
cluding hydroxylation, alkene epoxidation, O-dealkylation, and
sulfoxidation. P450LaMO thus possesses great potential as a bio-
catalyst for further directed evolution and diverse applications.
Spectral characterization, activity detection, and kinetics assays:
UV absorption spectra (400–500 nm) of the CO-bound recombinant
CYP proteins were measured after sodium dithionite reduction.
P450 monooxygenase concentrations were estimated from differ-
ence spectra between the reduced, CO-bound form and the re-
duced form, as described by Omura and Sato.[10] P450 monooxyge-
nase concentrations were then determined by use of an extinction
coefficient of 91 mmÀ1 cmÀ1 at 450 nm.
NADPH oxidation activities and kinetics assays were recorded at
308C with a Beckman DU 730 spectrophotometer (Beckman). Activ-
ities were determined by monitoring the decreases in absorbance
of NADPH at 340 nm (eNADPH =6.22 mmÀ1 cmÀ1) at 308C. The stan-
dard assay mixture (1 mL) consisted of p-chlorothioanisole
(0.2 mm) and P450LaMO (0.1–0.2 mm) in Tris·HCl buffer (pH 8.5,
50 mm). The reaction was initiated by the addition of NADPH
(0.1 mm). One unit of enzyme activity was defined as the amount
of enzyme catalyzing the oxidation of 1 mmol NADPH per minute.
For the reference sample, NADPH consumption rates were mea-
sured in the absence of substrate.
Experimental Section
General: d-Aminolevulinic acid and sodium dithionite were pur-
chased from Aladdin Chemicals Co., Ltd. (Shanghai, China). Cyto-
chrome c was purchased from Sigma–Aldrich. 7-Ethoxycoumarin
and 7-hydroxycoumarin were obtained from TCI (Shanghai, China).
All other chemicals used are widely commercially available and
were of analytical grade purity or higher.
Kits for plasmid extraction and DNA purification were purchased
from Qiagen (Shanghai, China). Restriction endonucleases and plas-
mid pMD18-T for the cloning of PCR products were obtained from
Takara (Dalian, China). Plasmid pET-28a(+) for heterogeneous ex-
pression was obtained from Novagen (Shanghai, China). The micro-
bial strains used for genome mining were obtained from the
German Collection of Microorganisms and Cell Cultures (DSMZ),
the National Collections of Industrial, Food and Marine Bacteria
(NCIMB Ltd; Aberdeen, UK), or from our own collection. E. coli
DH5a and E. coli BL21(DE3) used for cloning and expression of
P450 monooxygenases were from our own laboratory stocks.
Reductase activities were determined by a standard cytochrome c
assay. Each reaction was performed in Tris·HCl buffer (pH 8.5,
50 mm) containing P450LaMO (2 nm) and cytochrome c (0.1–0.4 mm).
The reaction was initiated by addition of NADPH (0.1 mm). Kinetic
constants, KM and kcat, were calculated by fitting the reaction rates
measured across a range of substrate concentrations to the Mi-
chaelis–Menten kinetic model.
Biotransformation mediated by P450LaMO: Reactions were per-
formed with NADPH cofactor regeneration by glucose dehydro-
genase (GDH) from Bacillus subtilis. The reaction mixtures (1 mL)
each contained substrate (500 mm, 1.0–2.0 mm for sulfide sub-
strates), NADP+ (200 mm), glucose (10 mm), and GDH (5 U) in
Tris·HCl buffer (pH 8.5, 100 mm). Each reaction was initiated by the
Genome data mining for CYP116B monooxygenases: A library of
putative monooxygenases was constructed by genome mining. A
total of 20 monooxygenases, each with 33–97% amino acid se-
quence homology to the templates P450SMO and P450RhF, were se-
lected from the UniProt/Swiss-Prot database. After heterogenous
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Yin, Yue-Cai
