CHAPTER 3 IN SILICO ANALYSIS OF SBP……………………………….. 40-72
3.3 RESULTS AND DISCUSSION
3.3.5 Cyclo/Maltodextrin ABC transporter
In T. thermophilus HB27, uptake of maltotriose is mediated by a maltose ABC transporter (ORF IDs: TTC1286-TTC1288) (Cuneo et al., 2009a). Genome sequence analysis of T.
thermophilus HB8 reveals the presence of a similar maltose ABC transporter (ORF IDs:
TTHA1650-TTHA1652). In fact, the SBP components (ORF IDs: TTC1288 and TTHA1652) of both the transporters are identical at the primary structure level (sequence identity: 99%, query coverage: 100%). Historically, maltose ABC transporters are known to import maltose and α(1→4)-linked glucose polymers (up to seven glucose units) such as maltodextrin (Boos and Shuman, 1998). Thus, in this study, we investigated the binding of glucose polymers such as maltodextrin (or cyclodextrin) to the protein TTHA1652.
The protein TTHA1652 can bind cyclo/maltodextrin molecules
In UniProtKB database, the protein TTHA1652 is annotated as “maltose ABC transporter, periplasmic maltose-binding protein” which shares the highest sequence similarity with maltose/maltodextrin-binding protein (ORF ID: TTC1288, PDB ID: 2GH9) from T.
thermophilus HB27. To infer the probable ligands in addition to maltose, tertiary structure of TTHA1652 was predicted using different programs. Expectedly, all programs used maltose/maltodextrin-binding protein (PDB ID: 2GH9, maltotriose bound and close conformation) as the default template, hinting maltotriose as the cognate substrate for the protein TTHA1652. Topologically, the protein TTHA1652 is homologous to maltose/maltodextrin-binding protein from T. thermophilus HB27 (PDB ID: 2GH9, maltotriose bound) and T. maritima (PDB IDs: 2GHA and 2FNC, maltotriose bound) and maltodextrin-binding protein from Thermoactinomyces vulgaris (TvuCMBP, PDB ID:
2ZYM, cyclodextrin bound) (Table A.2). The protein TvuCMBP binds glucose polymers such as maltodextrin e.g. maltotetraose (G4: 4 glucose units, PDB ID: 2ZYO) and
cyclodextrin (CD) e.g. α-CD (G6: 6 glucose units, PDB ID: 2ZYM), β-CD (G7: 7 glucose units, PDB ID: 2ZYN) and γ-CD (G8: 8 glucose units, PDB ID: 2ZYK) (Tonozuka et al., 2007; Matsumoto et al., 2009). Thus, the protein TTHA1652, which shares a similar topology to that of TvuCMBP, can be speculated to be capable of binding glucose polymers such as cyclo- and malto-dextrin molecules. Furthermore, the residues Leu59, Asp83, Glu129, Tyr175, Asn247, Trp250 and Trp360 of TvuCMBP, which holds the CD molecule in the active site of the protein, are conserved in the protein TTHA1652 as well (Figure 3.4A); suggesting cyclo/maltodextrin as its probable substrate.
Furthermore, to evaluate the binding of cyclo/maltodextrin to the protein TTHA1652, molecular docking experiments with maltose, maltodextrins and cyclodextrin were performed. The results suggest the binding of maltose in subsites either A-B or B-C (Figure A.5A, A.5B) while the smallest maltodextrin i.e. maltotriose and maltotetraose occupy subsites A-C and A-D, respectively (Figure A.5C, A.5D). Interestingly, among all the docked substrates, maltotriose and maltotetraose seems to be the preferred cognate substrates for the protein TTHA1652 (Table A.3). The larger docked molecules are stabilized mostly by stacking forces and few water molecules in the active site of the protein (Figure 3.4B). Thus, it can be suggested that the protein TTHA1652 binds and mediates the transport of maltodextrin molecules preferably up to 4-5 pyranose units.
As T. thermophilus HB8 contains cyclo/maltodextrin-metabolizing enzyme maltodextrin glucosidase (MalZ, ORF ID: TTHA1647), which catalyzes the hydrolysis of both maltodextrin and cyclodextrin (only γ-CD and not α- and β-CDs) molecules into glucose and maltose (Boos and Shuman, 1998) and also the homologous protein TvuCMBP transports cyclo/maltodextrin molecules, the transport of γ-CD in addition to maltodextrin can be extrapolated for the protein TTHA1652 as well. To affirm this, we performed molecular docking of TTHA1652 and TvuCMBP (as control) with γ-CD as the ligand. The results demonstrate that like TvuCMBP, the protein TTHA1652 can accommodate γ-CD in its active site with the help of the conserved residues Phe39, Tyr157, Trp235 and Trp345, which stack the G1c2, Glc3 and Glc4 units of γ-CD (Figure 3.4C and Table A.3).
These results were further substantiated by investigating the genetic machineries associated with cyclo/maltodextrin transport and metabolism in T. thermophilus HB8, HB27 and T.
maritima (Figure 3.4D). The presence of functionally-associated cyclo/maltodextrin- metabolizing enzymes upstream to the ABC transport system suggests that in T.
thermophilus HB8, the import of the cyclo/maltodextrin is performed by the TTHA1650- TTHA1652 ABC transporter for its further metabolism via the enzyme MalZ (ORF ID:
TTHA1647). Subsequently, the metabolized products of cyclo/maltodextrins such as glucose and maltose enter the glycolysis pathway for downstream processing (Figure A.6).
In T. thermophilus HB8, the presence of the enzyme neopullulanase (PDB ID: 2Z1K), which hydrolyses glycosidic linkages (α-1,4 and α-1,6) of pullulan and cyclodextrins (Cheong et al., 2002; Lee et al., 2002), further corroborates our hypothesis.
Figure 3.4. Active site and genetic organization of TTHA1652. (A) Multiple sequence alignment of TTHA1652_Tt (T. thermophilus HB8) along with 2ZYM_CMBP_Tv (T.
vulgaris), 2GH9_MBP_Tt (T. thermophilus HB27), 2GHA_MBP_Tm (T. maritima), 2FNC_MBP_Tm (T. maritima) and 1URD_MMBP_Aa (Alicyclobacillus acidocaldarius).
For the clarity of the figure, only partial alignment is shown. The amino acid residues involved in interaction with G1c2, Glc3 and Glc4 of CD are shaded in green and
highlighted with green downward arrowheads. The hydrophobic residues interacting with the central cavity of CD and maltotetraose are highlighted in cyan and blue downward arrowheads. (B) Superimposition of the active-site pocket of maltotetraose-binding protein (PDB ID: 3K00, grey) and the protein TTHA1652 (cyan). The maltotetraose bound to the crystal structure (PDB ID: 3K00) and that obtained from the molecular docking calculation is presented as ball-and-stick model in black and green, respectively. The aromatic residues of TTHA1652 essential for the binding of maltotetraose are shown in cyan. (C) The comparison of γ-CD binding in TvuCMBP (grey) and TTHA1652 (cyan). The positioning of γ-CD from the crystal structure (PDB ID: 2ZYK) and the molecular docking experiment is shown in black and green, respectively. The aromatic residues Phe9, Phe39, Tyr157, Tyr215, Trp235 and Trp345 of TTHA1652 holding the G1c2, Glc3 and Glc4 units of γ- CD are depicted in cyan. (D) Schematic representation of ORFs for maltodextrin transport and metabolism systems in T. thermophilus HB8 (upper) and HB27 (lower) and T.
maritima (PDB IDs: 2GHA (upper) and 2FNC (lower)). Each gene is represented by an arrow indicating the direction of transcription along with its ORF number and respective encoded protein names. ORFs encoding a functionally similar protein across the organisms are depicted in similar color such as ABC transporter, green; maltodextrin glucosidase, cyan; transcription regulator, magenta; endo-1,4-β-galactosidase, blue; cyclo- maltodextrinase, navy blue; α-amylase, purple and pullulanase, yellow.