Amino Acid Sequence of Amylase Type Alpha, MiAmy, from Ok-Rong Mango (Mangifera indica Linn. cv. Ok-Rong)

1Department of Biochemistry, Faculty of Science, Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, 40002, Thailand 2Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand 3Chemistry Program, Faculty of Science and Technology, Nakhon Ratchasima Rajabhat University, Nakhon Ratchasima, 30000, Thailand


Introduction
α Amylases are classified into amylase family-13 (EC 3.2.1.1) or GH-13 which hydrolyzes α-1, 4-Dglucosidic bonds. These proteins are found in diverse organisms and are still being characterized. α Amylases can be utilized for diverse applications, such as in the detergent, textile, paper, sugar and ethanol industries. For each application, it is important to understand the enzyme's specificity and function. Recently, a large number of α amylase proteins have been discovered in sources such as bacteria, fungi, cereals, mammal and higher plants including fruits (Torgerson et al., 1979;Kondo et al., 1980;Robyt and French, 1970;MacGregor and MacGregor, 1985;Stanley et al., 2002;SWISS-PROT, 2000;GenBank, 1982). These newly discovered enzymes vary in their structures and specificity.
Ok-rong mango (Mangifera indica Linn. cv. Okrong) is a plant indigenous to Thailand (local name; Mamuang Ok-rong). It is a large green tree that grows up to 20 m tall. Mangos thrive in both the subtropics and the tropics and are one of the most popular fruits both in ripe and green stages. The ripening process involves changes to several biochemical compounds which in turn cause changes in color, flavor, texture and taste. For example, in regard to flavor, soluble sugars, account for mango sweetness and accumulate through carbon supplied during both photosynthesis and starch degradation, which occur during ripening. During fruit development, starch accumulates up to 8% in the fresh pulp weight, but a low amount of soluble sugars is detected. However, during ripening, accumulated starch is rapidly converted into soluble sugars, which that can reach as high as 10% of the fresh pulp weight (Peroni Goncalves et al., 2008).
In previous studies, the amylase activity during ripening of Ok-rong mangos was significantly higher than in 30 other tested fruits (personal communication). Therefore, it is possible to discover an isoform of αamylase that has a high specific activity, many favorable properties and a high potential for development for industrial applications.

Plant Materials
Ok-rong mangos (Mangifera indica) in the ripening stage of were collected from Srisaket Province in northeastern Thailand. Pulp was ground under liquid nitrogen and stored at -80°C before use.

Amino Acid Sequence Determination
Ripening Ok-rong mango tissues were disrupted by the addition of liquid nitrogen and subsequent homogenization. Total RNA was extracted using TRIzol® reagent. Then, cDNA sample were synthesized using the cDNA synthesis kit (ThermoScript™). For α amylase gene amplification, primers (Table 1) were designed from the α amylase sequences from other plants, Arabidopsis thaliana; AY065233, Malus domestica; AAF63939 and Citrus sinensis; XP_006483229 (Genbank). Next, specific primers were designed from our α amylase sequence obtained above. For PCR reactions, pre-denaturation was carried out at 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 2 min. A final extension step at 72°C was conducted for 10 min. The amplicons were purified and ligated into the pGEM-T Easy vector (Promega, USA). DNA plasmids were purified and sequenced (1st Base Company, Malaysia).
The 3 ′ RACE system was carried out according to the kit's instruction manual (Invitrogen, USA) using the AUAP universal primer as a reverse primer. The PCR conditions were to mix sample in the green PCR master mix kit (Fermentas, Singapore) and thermocycle at 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 min and final extension at 72°C for 10 min. Then, the amplicons were purified and ligated into the pGEM-T Easy vector (Promega, USA). DNA plasmid were purified and sequenced.

MiAmy Amino Acid Sequence Determination
Five nucleotide fragments were obtained from RT-PCR techniques using 5 pairs of primers. The 3 ′ end was determined using 3 ′ RACE. There were completely overlapped (Fig. 1). Six sequence fragments were found to be part of the α amylase sequence. The sequence was found to be 1782 base pairs of nucleotides and share 85, 83 and 79% identity with α amylase from durian (D. zibethinus cv. Mon thong), sweet orange (C. sinensis), mulberry (M. notabilis), respectively. The nucleotide sequence encoded 594 amino acid residues which shared 78 and 77% identity with chloroplastid α amylase 3, from D. zibethinus and C. sinensis, respectively. It was named MiAmy.

Multiple Alignment for Sequence Similarities
Well-described sequences from previous studies (Nakajima et al., 1986;Janecek, 1992) were inputs into the CLUSTALW program and used to generate amino acid sequence alignments for α-amylase. The large Nterminal domain of plastid α-amylase, amino acids 447-460, was removed. The pre-defined sequences contained four conserved and semi-conserved positions, including the active site, which allowed similarities to be identified for the same regions in the novel sequence. The alignments revealed high conservation between sequence with substitutions by similar amino acid residues when compared with those from other sources including plants, mammals and bacteria. The four conserved regions are region (I) DAVLNH, (II) GWRLDFVRG, (III) GEYWD and (IV) FIENHDT. From structural studies, region II and III are suggested to be specific for the α-1, 4 glucosidic bond for α amylase family 13. The study of their anomeric configuration revealed their activity at one end of a polysaccharide (MacGregor et al., 2001). The four highly conserved residues are the substrate binding site; H291 and the catalytic sites; D372, E397 and D481 presented in conserved region I, II, III and IV, respectively (Fig. 2).

Evolution Tree of α Amylase Ok-Rong Mango
Phylogenetic trees with unrooted distances were calculated based on the Neighbor-Joining method (Saitou and Nei, 1987) based on the sequence similarities alignment (Fig. 2). The MiAmy was grouped into clusters along with the plastid α amylase members (Fig. 3). The tree reflects data from previous studies, such as a difference between conserved amino acid residues in the conserved region, especially region four. For grouping of only plastid α amylases, MiAmy was branched nearest to DzAmyF3 (D. zibethinus cv. Mon Thong) and CsAmyF3 (C. sinensis). This evolutionary study corresponds to the high (78 and 77%) similarity of alignment confirming that MiAmy is an α amylase from plastids.  Table 2. Amino acids 447-460 of plastid α-amylases were removed. The four well-accepted regions I, II, III and IV are surrounded by rectangles. The conserved binding site and active site residues are numbered H291, D372, E397 and D481, which are present in the 4 well-accepted conserved regions I, II, III and IV, respectively. The conserved residues that are thought to be a part of the Ca 2+ binding site are labeled with "C." The residues involved in hydrogen bonding to the α-amylase inhibitor acarbose are conserved and labeled "Z." A predicted sugar tong binding site in domain C is labeled "S." The names and identifying details for α-amylases are shown in Table 2 Fig and C (blue) containing D372, E397 and D481 as active site residues using HvAmyF1 (1RP8) as a template. B: A merged structure of α-amylase from Ok-rong mango (color) and template (gray); the structure displayed shows the sugar tong binding site and starch granule binding site as gray sticks α Amylase Secondary Structure Prediction InterProScan-an integration platform for the signature-recognition methods in InterPro Bioinformatics was used to determine the positions of helices and extended-chains along the polypeptide chains of plants α amylase. The comparison of secondary structure between MiAmy and the well study HvAmyF1 confirmed that 594 aa contained with active Domain A, substrate binding Domain B and Cterminus Domain C (Fig. 4).

α Amylase Three Dimensional Structure Prediction
For structural grouping, the predicted 3D structure of α-amylase from Ok-rong mango was generated using the SPDBV Swiss model program and Pymol using 1RP8.pdb (crystal structure of barley alpha-amylase isoform 1) as a template (Fig. 5). The partial amino acid sequence of Ok-rong mango α-amylase displayed 42% sequence identity with the X-ray structure of barley αamylase and Domain A was conserved.

Discussion
In this study, 594-amino acid MiAmy with an active domain, was confirmed to be a plastid α amylase and classified into the family 13. The alignment revealed the difference between secretory α amylases and plastid α amylases. In the fourth region, the protein can be classified into two groups, FVDNHD and FIENHD for secretory and plastid α amylases, respectively. MiAmy region four allowed the protein to be classified into the group of plastid α amylase. However, the difference between VD and IE in this region does not affect the mechanistic properties. In this study, nearly all critical domains and region of MiAmy are found, which are as follows: (1) the two consecutive tryptophan residues for starch-granule binding, which correspond to W469 and W470 (Tangphatsornruang et al., 2005), (2) the conserved residues thought to be the Ca 2+ binding site, (3) the residues involved in hydrogen bonding with the αamylase inhibitor acarbose, (4) Tyr 570 in domain C, corresponding to Tyr807 of DzAmyF3, which was suggested to be a sugar tongs surface binding site (Posoongnoen et al., 2015). Thus, the MiAmy αamylase gene is predicted to be valuable for further studies, including its modification.
The secondary structure of the catalytic domain of all members, including MiAmy showed the same basic (β/α) 8 barrel, eight β stands surrounded by eight α helices.  2). This result correspond with the first 380 residues of secretory Taka-amylase A which constitutes the active domain necessary for α-amylase activity (Matsuura et al., 1984). For chloroplastid α-amylase, the active domain was characterized as the middle to the C-terminal end. Previous studies reported that an active domain, comprising amino acids 380-440, is necessary for activity of α-amylase (MacGregor, 1988). At least 101 amino acid residues of the C-terminus could be removed without the losing enzyme activity (Yamazaki et al., 1983).

Conclusion
An amino acid sequence of α-amylase from Okrong mango (Mangifera indica), MiAmy, was determined and classified as a plastid amylase. Bioinformatics revealed that only the amino acid residues forming the active domain are important for function and activity. In support of a previous report, 496 aa at the N-terminus of AtAmy3 was removed. The construction of recombinant shortened (390-aa) AtAmy3 with an active domain showed equal activity when compared with the full-length native 887-aa AtAmy3 (Yu et al., 2005), which supports that the identified sequence for MiAmy, containing the active domain, is sufficient for cloning and expression.