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L.S. Sarker et al. / Archives of Biochemistry and Biophysics 528 (2012) 163–170
specific primer sets containing appropriate restriction enzyme
sites (Table 1) for cloning in an expression vector. The Amplicons
were digested with Nde I and EcoR I/Xho I restriction enzymes
and ligated into pET41b(+) bacterial expression vector where it
was fused to sequences encoding eight C-terminus Histidine resi-
dues to facilitate purification by Ni–NTA agarose affinity chroma-
tography (EMD Chemicals, Darmstadt, Germany). To produce the
recombinant protein, Escherichia coli Rosetta™ (DE3)plysS cells
(EMD Chemicals, Darmstadt, Germany) were transformed with
individual constructs, grown to log phase at 20 °C in Luria–Bertani
O
OPP
HO
BDH
Borneol
Camphor
GPP
Fig. 1. Proposed pathway for camphor biosynthesis. Multiple arrows indicate
involvement of multiple enzymes. GPP, geranyl diphosphate; OPP, diphosphate
moiety; BDH, borneol dehydrogenase.
(
LB) media supplemented with 30 mg/L Kanamycin and 34 mg/L
chloramphenicol, and induced with isopropyl-ß- -thiogalactopy-
D
C10 precursor to all regular monoterpenes, which is subsequently
ranoside (IPTG) at a final concentration of 0.5 mM. The induced
cells were chilled on ice for 15–20 min, collected by centrifugation
at 3,220g and 4 °C for 20 min, and stored at ꢁ80 °C overnight. The
stored cells were resuspended in Novagen bind buffer (0.3 M NaCl,
cyclized and hydrolyzed to borneol through the catalytic activities
of bornyl diphosphate synthases and borneol synthases, respec-
tively [13,14]. Oxidation of borneol will then generate camphor
through the catalytic activity of borneol dehydrogenase (BDH)
5
2 4
0 mM Na HPO , 10 mM imidazole, pH 8.0; EMD Chemicals, Ger-
[
10,15]. So far a specific plant BDH has not been reported. However,
many) containing 1 mM protease inhibitor phenylmethanesulfonyl
fluoride (PMSF), and sonicated on ice using a Sonic Dismembrator
Model 100 (Fisher Scientific, Ottawa, ON, Canada) to complete bac-
terial membrane disruption. The cell debris were removed by cen-
trifugation at 15,000g and 4 °C for 15 min (Sorvall, USA), and the
recombinant proteins harvested from the soluble fraction by
Ni–NTA agarose affinity chromatography (EMD Chemicals,
Germany) following the manufacturer’s procedure. Protein
samples were resolved by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS–PAGE) and visualized by staining with
Coomassie Brilliant Blue. Protein concentration was determined
by Bradford Assay (Bio-Rad).
a non-specific (accepting multiple substrates) short chain alcohol
dehydrogenase (SDR) from Artemisia annua was recently shown to
produce the corresponding ketones from a number of monoterpene
alcohols, including borneol, as a minor substrate [16].
Members of the SDR super-family of enzymes, which are found
in all living organisms, generally share low sequence similarity
level and have affinity towards structurally diverse substrates.
The C-terminal domains of SDRs determine substrate specificity
and can be highly variable between different SDR members
[
17,18]. The only common characteristic feature of all SDRs are
their short size (about 250 amino acids), the co-factor binding
Rossmann-fold scaffold, and their ability to bind NAD(P)(H). The
Rossmann-fold scaffold is characterized by a twisted parallel
Initially, enzyme assays were performed in 5 ml of 100 mM so-
dium phosphate buffer [12,23,24] (pH 8.0), containing 40 lg of the
b-sheet flanked on either side by 3–4
sified into five sub-families denoted as ‘‘classical’’, ‘‘extended’’,
‘intermediate’’, ‘‘complex’’, and ‘‘divergent’’ according to sequence
a-helices [19]. SDRs are clas-
+
enzyme, 1 mM NAD , and 0.5 mM substrate (borneol). After over-
night incubation at 30 °C with 150 r.p.m shaking, assay products
were extracted into 1 ml pentane and concentrated ꢂ50 times be-
fore analysis by GC–MS (see below). For linear kinetics study, as-
says were performed in 2 ml reaction volume (keeping reagent
concentrations as before) at five different time points: 1, 2, 4, 8,
and 16 h. The optimum temperature was determined from a set
of reactions performed at 27, 30, 32, 35 and 37 °C. The optimum
pH was determined by performing assays at pH 7.0, 7.5, 8.0, 8.5,
‘
combinations in their conserved motifs residing in the cofactor
binding and active sites [17,20]. The conserved motifs that define
the classical SDRs sub-family members are TGxxx[AG]xG and
YxxxK. The Gly rich motif in the cofactor-binding site determines
+
+
the protein’s cofactor (NAD or NADP ) specificity, while adjacent
serine and lysine residues flank the tyrosine based catalytic center
[
17]. Tyr is a catalytic residue, and Lys has a dual function as it
9
.0 and 10.0 using MOPS for pH 7.0 and pH 7.5, sodium phosphate
a
interacts with the coenzyme and lowers the pK value of the Tyr
through a strong electrostatic influence [21].
for pH 8.0, TAPS for pH 8.5, and CAPSO for pH 9.0 and 10.0 as a
buffer, respectively. All assays were performed in duplicate or
triplicate.
To construct the Michaelis–Menten saturation curve, enzyme as-
says (n = 5) were performed at optimum temperature (32 °C) and pH
SDR enzymes play important roles in the metabolism of lipids,
proteins, and carbohydrates. They have also demonstrated roles
in specialized metabolism in plants [10,17]. Recently, a few SDRs
have been reported from A. annua and Zingiber zerumbet, where
they are responsible for the biosynthesis of mono- and sesquiterp-
enoid ketones from multiple substrates [10,16]. In this study, we
employed a homology-based cloning strategy to clone a SDR from
L. x intermedia oil gland library (LiBDH), which converts borneol
into camphor in vitro.
(
8.0) for 30 min in 1 ml reaction volume containing 100 mM sodium
+
phosphate buffer, 1
tration of 5, 25, 50, 100, 200, 400
l
M enzyme, 1 mM NAD , and substrate concen-
M and 1 mM. Assay progress was
l
+
monitored by measuring the conversion of NAD to NADH at 340 nm
using a Lambda 25 UV–visible spectrometer (Perkin-Elmer). The
kinetic parameters of the enzyme were determined from a Michae-
lis–Menten saturation curve constructed using SigmaPlot software
version v.10.00 (Systat Software, Germany).
Material and methods
EST database analysis and BDH candidate selection
Table 1
We have recently reported the construction of a cDNA library
and the corresponding EST database for the floral glandular tric-
homes of mature (30% in bloom) L. x intermedia flowers [22]. Based
on homology to known SDRs, four full-length BDH candidates were
selected and fully sequenced prior to further analysis.
Oligonucleotides used in this study.
Primer type
Full length
Target gene
LiBDH
Primers
0
0
0
0
0
0
0
0
0
F-5 -CCCTCATATGGCTTCAACTGTTTTGAGA-3
0
R-5 -AGTCTCGAGCGAATCCATCAAATCAAAC-3
0
0
qPCR
LiBDH
LiLINS
b-actin
F-5 -AATCGGAGCGGCAGCATAATCT-3
R-5 -TAATACGGCGAGACGCAGTTCA-3
0
Recombinant protein expression and enzyme assay
F-5 -ACACGCACGACAATTTGCCA-3
0
0
R-5 -AGCCCTCCAATGAAGTGGGAT-3
F-5 -TGTGGATTGCCAAGGCAGAGT-3
The predicted ORFs for BDH candidates were amplified by PCR
using iProof high fidelity DNA polymerase (Bio-Rad, USA) and
0
R-5 -AATGAGCAGGCAGCAACAGCA-3
Sarker, Lukman S.
