Total RNA preparation from T. camphorata and cDNA synthesis
Fruiting bodies of T. camphorata, a fungus that occurs only in the inner cavity of an endangered tree Cinnamonum kanehirai, were obtained from Asian Nova Biotechnology Inc, Taiwan (http://www.asian-bio.com/). Fresh fruiting bodies (wet weight 10 g) were frozen in liquid nitrogen and ground to powder in a ceramic mortar. PolyA mRNA (25 μg) was prepared using Novagen’s Straight A’s mRNA Isolation System (Gibbstown, NJ, USA). Four micrograms of the mRNA were used in the 5′-RACE-Ready cDNA and 3′-RACE-Ready cDNA synthesis using Clontech’s SMART RACE cDNA Amplification Kit (Mountain View, CA).
Isolation of TcAAD cDNA
We have previously established an EST database from fruiting bodies of T. camphorata and sequenced all clones with insert size greater than 0.4 kb (data not shown). The identity of a partial AAD cDNA clone was assigned by comparing the inferred amino acid sequence in various databases using the basic local alignment search tool (BLAST) (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Using the T. camphorata 5′-RACE-Ready cDNA as a template, an UPM primer (universal primer A mix, purchased from BD Biosciences, Palo Alto, CA) and a primer (5′GAT ATT CCA CTC GGC CTT GAC C3′ ) based on AAD partial cDNA, a 700 bp fragment was amplified by PCR. The 700 bp fragment was subsequently subcloned and sequenced. Based on the DNA sequence, a reverse primer AAD-1 (5′ CCT CTC GAG GGA GAT TCA A 3′) and a forward primer AAD-2 (5′GAG GTG ATG CTG GGG AAT3′) were synthesized. Using the T. camphorata 5′-RACE-Ready cDNA as a template and AAD-1 and UPM primer pair, a 400 bp fragment was amplified by PCR. Using the T. camphorata 3′-RACE-Ready cDNA as a template and AAD-2 and UPM primer pair, a 1,200 bp fragment was amplified by PCR. Both DNA fragments were subcloned into pCR4.0 vector and transformed into E. coli TOPO10, separately. The nucleotide sequence of the inserts was determined in both strands. Sequence analysis revealed that the combined sequences (400, 700 and 1,200 bp) covered an open reading frame of a putative AAD cDNA (1299 bp, GenBank no. HQ453361). The identity of this TcAAD clone was assigned by comparing the inferred amino acid sequence in various databases using the basic local alignment search tool (BLAST).
Bioinformatics analysis of TcAAD
The BLAST program was used to search homologous protein sequences in the nonredundant database (NRDB) at the National Center for Biotechnology Information, National Institutes of Health (http://www.ncbi.nlm.nih.gov/). Multiple alignments were constructed using ClustalW2 program. Protein secondary structure was predicted by SWISS-MODEL program and represented as α helices and β strands. A 3-D structural model of TcAAD was created by SWISS-MODEL (Arnold et al., 2006) (http://swissmodel.expasy.org/) based on the known crystal structure of voltage-dependent potassium channels (PDB code:3EAU).
Subcloning of TcAAD cDNA into an E. coli and yeast expression vector
The coding region of the TcAAD cDNA was amplified using gene specific flanking primers. The 5′ upstream primer contains Eco RI recognition site (5′GAA TTC GAT GTC CGT GGA GAA GAA GTC3′) and the 3′ downstream primer contains Hind III recognition site (5′AAG CTT AGA ATG GCC ATC GGC CAA C 3′). Using 0.2 μg of TcAAD cDNA as a template, and 10 pmole of each 5′ upstream and 3′ downstream primers, a 1,044 bp fragment encoding the putative mature TcAAD gene was amplified by PCR, and was ligated with cloning vector pCR4.0 and transformed into E. coli. The plasmid was isolated and digested with Eco RI and Hind III. The digestion products were separated on a 1.0% agarose gel. The 1,044 bp insert DNA was gel purified and subcloned into Eco RI and Hind III site of pET-20b(+) expression vector (Novagen, Darmstadt, Germany). The recombinant DNA was then transformed into E. coli C43(DE3). The recombinant protein was not overexpressed in the E. coli expression system.
Therefore the TcAAD gene was subcloned into a yeast expression system for overexpression. The coding region of the TcAAD cDNA was re-amplified by using two gene-specific primers: the 5′ upstream primer contains Eco RI recognition site (5′GAA TTC GAT GTC CGT GGA GAA GAA GTC3′) whereas the 3′ downstream primer contained a His6-tag and Eco RI recognition site (5′ CGT CTC GAA TTC TCA GTG GTG GTG GTG GTG GTG 3′). Using the 0.2 μg recombinant DNA of pET-20b(+)-TcAAD as a template, and 10 pmole of each 5′ upstream and 3′ downstream primers, a 1.0 kb fragment was amplified by PCR. The fragment was ligated into pCR4.0 and transformed into E. coli. The recombinant plasmid was isolated and digested with Eco RI. The digestion products were separated on a 1.0% agarose gel. The 1.0 kb insert DNA was gel purified and subcloned into the Eco RI site of the pYEX-S1 expression vector (Clontech, Mountain View, CA, USA) and introduced into Saccharomyces cerevisiae (trp- ura-). The transformed yeast cells were selected by YNBDT (0.17% yeast nitrogen base, 0.5% ammonium sulfate, and 2% glucose) agar plates containing 20 μg Trp/mL. The presence of TcAAD gene in the selected transformants was verified by PCR using gene-specific flanking primers. The recombinant TcAAD protein was expressed in yeast in YPD medium (1% yeast extract, 2% peptone, 2% glucose). Overexpression of the functional recombinant TcAAD was analyzed by enzyme activity assay.
Expression and purification of the recombinant TcAAD
The yeast transformant which containing the TcAAD gene was grown at 30°C, 170 rpm in 250 mL of YPD medium for 2 days. The cells were harvested and the soluble proteins extracted in PBS (phosphate buffer saline) with glass beads as described previously (Ken et al., 2006). The recombinant TcAAD was purified by Ni-NTA affinity chromatography (elution buffer: 30% PBS containing 20–250 mM imidazole) according to manufacturer’s instruction (Qiagen). The purified protein was analyzed by a 12% SDS-PAGE followed by staining with Coomassie Brilliant Blue R-250 and destaining. Protein concentration was determined by a Bio-Rad Protein Assay Kit (Richmond, CA) using bovine serum albumin as a standard (Bradford, 1976).
Molecular mass analysis via JOEL MALDI-TOF (JMS-S3000, Japan)
The purified recombinant TcAAD (1 mg/mL) was dissolved in 0.3% PBS containing 0.05 mM imidazole and 0.45% glycerol. The sample (5 μL) was used for molecular mass determination using JOEL MALDI-TOF.
TcAAD activity assay
The AAD activity was determined by measuring NADPH-dependent reduction of veratraldehyde (3,4-dimethoxybenzaldehyde, (CH3O)2C6H3CHO) at pH 6.0 (Muheim et al., 1991; Guillen and Evans, 1994). A typical 100 μL reaction mixture contained 25 mM bis-tris- propane/HCl (pH 6.0), 0.2 mM NADPH and 0.2 mM veratraldehyde. The reaction was initiated by addition of 3 μg TcAAD. The reaction was followed by a decrease in A365 due to the oxidation of NADPH. A365 was used instead of A340 to reduce the interferences with the maximum absorbance of veratraldehyde at A310 (Guillen and Evans, 1994). Under the same conditions, another set of reactions was set up except that NADPH was replaced with NADH for enzyme activity assay. The molar absorption coefficient of NADH at 355 nm is 4390 M-1 cm-1.
The ability of the TcAAD to oxidize benzyl alcohols (benzyl alcohol; 2,4-dimethoxy benzyl alcohol; 3,4-dimethoxybenzyl alcohol and 4-(hydroxymethyl)benzoic acid) was tested by measuring NAD+-dependent oxidation of these benzyl alcohols to their corresponding benzylaldehydes by increasing the production of NADH at A355 nm. A typical 100 μL reaction mixture contained 50 mM glycine/NaOH (pH 9.6), 4 mM NAD+ and 4 mM benzyl alcohols (Siljegovic et al., 1998). The reaction was initiated by addition of 10 μg TcAAD. The reaction was followed by an increase in A355 due to the reduction of NAD+. Under the same conditions, another set of reactions was set up except for NAD+ was replaced with NADP+ for enzyme activity assay.
The kinetic properties of the TcAAD (3 μg) was determined by varying the concentrations of veratraldehyde (0.1 to 0.4 mM) with fixed amount of 0.2 mM NADPH. The change in absorbance at 365 nm was recorded for one min. The molar absorption coefficient of NADPH at 365 nm is 3.5 mM-1 cm-1. The KM, Vmax and kcat were calculated from Lineweaver-Burk plots.
The TcAAD enzyme was tested for stability in terms of its activity under various conditions. Aliquots of the TcAAD sample were treated as follows: (1) Thermal effect. Each enzyme sample (3 μg/12 μL) was heated to 58°C for 2, 4, 8 or 16 min. Temperatures which are greater than 58°C were also performed. (2) pH effect. Each enzyme sample (3 μg/12 μL) was adjusted to desired pH by adding a half volume of buffer with different pHs: 0.2 M citrate buffer (pH 4.0), 0.2 M phosphate buffer (pH 5.0, 6.0, 7.0, 8.0, or 9.0) or 0.2 M CAPS buffer (pH 10.0). Each sample was incubated at room temperature for 30 min. At the end of each treatment, TcAAD enzyme activity were checked at pH 6.0 in the presence NADPH or was subjected to 12% SDS-PAGE analysis as mentioned above.