Identification and characterization of MalA in the maltose/maltodextrin operon of Sulfolobus acidocaldarius DSM639

Abstract

A putative maltose/maltodextrin operon was found in the Sulfolobus acidocaldarius DSM639 genome. The gene cluster consisted of 7 genes (malA, trmB, amyA, malG, malF, malE, and malK). Here, we report the identification of MalA, which is responsible for the hydrolysis of maltose or maltodextrin to glucose in S. acidocaldarius. The transcription level of malA was increased 3-fold upon the addition of maltose or starch to the medium. Moreover, the α-glucosidase activity for maltose as a substrate in cell extracts of S. acidocaldarius DSM639 was also 11- and 10-fold higher during growth in YT medium (Brock's mineral salts, 0.1% [wt/vol] tryptone, and 0.005% [wt/vol] yeast extract) containing maltose or starch, respectively, than during growth on other sugars. The gene encoding MalA was cloned and expressed in S. acidocaldarius. The enzyme purified from the organism was a dodecamer in its active state and showed strong maltose-hydrolyzing activity at 100°C and pH 5.0. MalA was remarkably thermostable, with half-lives of 33.8 h, 10.6 h, and 1.8 h at 95°C, 100°C, and 105°C, respectively. Substrate specificity and kinetic studies of MalA with maltooligosaccharides indicated that MalA efficiently hydrolyzed maltose to maltopentaose, which is a typical characteristic of GH31-type α-glucosidases. However, glycogen or starch was not hydrolyzed. Reverse transcription-PCR, sugar uptake, and growth studies of the wild-type DSM639 and ΔmalEFG mutant on different sugars demonstrated that MalA located in the mal operon gene cluster is involved in maltose and starch metabolism in S. acidocaldarius.

Fig 1

RT-PCR and β-glycosidase activity analysis of the putative maltose/maltodextrin operon in S. acidocaldarius. (A) Putative maltose/maltodextrin operon gene cluster in S. acidocaldarius. (B) RT-PCR analyses of the genes in the operon associated with growth on maltose or starch. N, no sugar; G, glucose; M, maltose; St, starch; X, xylose; Su, sucrose; A, arabinose. The control was performed to identify PCR intensities of all primers against genomic DNA. Lane 1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH); lane 2, MalA; lane 3, TrmB; lane 4, AmyA; lane 5, MalG; lane 6, MalF; lane 7, MalE; lane 8, MalK. (C and D) β-Glycosidase activities expressed from the trmB promoter and malE promoter, respectively. S. acidocaldarius MR31 harboring pKHlac1 (pC::P_trmB-lacS) and MR31 harboring pKHlac2 (pC::P_malE-lacS), were grown in YT medium supplemented with 0.4% sugars. The levels of accumulated β-glycosidase activity were measured at exponential phase. Error bars represent the standard deviations of triplicate measurements.

Fig 2

Determination of MalA activity in S. acidocaldarius DSM639 (black bars) and the ΔmalEFG mutant (gray bars) grown in medium containing different sugars. No, no sugar; G, glucose; M, maltose; St, starch; X, xylose; Su, sucrose; A, arabinose.

Fig 3

Predicted primary structure of MalA expressed in S. acidocaldarius and its comparison with other related GH31 α-glucosidases. (A) Schematic representation of the primary structure of MalA expressed from pKHmalA. Gray boxes represent the amino acids (aa) added to the native MalA for successful expression and purification from S. acidocaldarius. Black boxes represent conserved regions in the GH31 amylolytic enzymes. (B) Comparison of MalA with other related GH31 α-glucosidases. The invariant sequences are highlighted in black with white characters, and the catalytic triads are indicated with asterisks. The highly conserved sequences are emphasized with boxes. Bth, Bacillus thermoamyloliquefaciens KP1071 α-glucosidase (BAA76396); Tth, Thermus thermophilus HB8 α-glucosidase (BAD71829); Afl, Aspergillus flavus NRRL 3357 α-glucosidase (EED47989); Sso, S. solfataricus P2 MalA (AAK43151); Tvo, Thermoplasma volcanium GSS1 TVN1302 (BAB60467); Tac, Thermoplasma acidophilum AglA (CAC11443); MalAsaci, S. acidocaldarius DSM639 α-glucosidase. Multiple alignments were performed using Clustal W2 and visualized using ESPript (http://espript.ibcp.fr/ESPript/ESPript/).

Fig 4

SDS-PAGE analysis of purified MalA and molecular mass determination. (A) SDS-PAGE analysis was performed at each purification step. Lane M, protein size standards; lane 1, cell extracts of MR31 harboring pC (36 μg); lane 2, cell extracts of MR31 harboring pKHmalA (28 μg); lane 3, soluble fraction proteins after sonication of MR31 harboring pKHmalA (28 μg); lane 4, proteins after Ni-NTA affinity chromatography (7 μg). (B) The molecular mass of the native MalA protein was estimated by gel filtration chromatography using a Sephacryl S-300 HR 16/60 column. The column was calibrated with the following molecular mass standards: thyroglobulin (670 kDa) (- · - · ); apoferritin (440 kDa) (---); β-amylase (200 kDa) (- · · -); and MalA (914 kDa) (—). mAu, milli-arbitrary units.

Fig 5

Effect of pH and temperature on the activity and stability of MalA. (A) For optimal pH determination, the following buffers were used: pH 3.0 to 4.5, sodium citrate (▼); pH 4.5 to 6.5, sodium acetate (○); and pH 6.5 to 8.0, HEPES (●). Activity was assayed at 95°C for 5 min in various buffers (50 mM). (B) To determine the optimal temperature, activity was measured at the indicated temperatures under the standard conditions of the assay. (C) To determine the thermostability of MalA, the enzyme was preincubated at 95°C (●), 100°C (■), and 105°C (▲) in 50 mM sodium acetate buffer (pH 5.0) without a substrate. After various time intervals, samples were withdrawn and the residual activity was measured at 95°C for 5 min using pNPG. Error bars represent the standard deviations of results from 3 separate experiments.

Fig 6

Growth of S. acidocaldarius DSM639 (●), MR31 (▲), and the ΔmalEFG mutant (▼) in NYT and YT media supplemented with different sugars. Growth was determined at 77°C, and 0.4% solutions of sugars were used. (A) YT medium without sugar; (B to G), YT medium containing glucose, xylose, sucrose, maltose, maltotriose, and starch, respectively; H and I, NYT medium containing maltotriose and starch, respectively.

Fig 7

Uptake of glucose (A), xylose (B), and maltose (C) by S. acidocaldarius DSM639 (●), MR31 (▲), and ΔmalEFG mutant (▼) cells grown on glucose, xylose, and maltose. The cells grown on 0.4% glucose, xylose, or maltose were incubated at 77°C with 5 mM glucose, xylose, or maltose, and the transported sugar was determined by HPLC in each case. The cell weights used were identical for the sugar transport assays.