Molecular and biochemical analyses of the GH44 module of CbMan5B/Cel44A, a bifunctional enzyme from the hyperthermophilic bacterium Caldicellulosiruptor bescii

Abstract

A large polypeptide encoded in the genome of the thermophilic bacterium Caldicellulosiruptor bescii was determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol. 78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases from C. bescii led to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization by C. bescii.

Fig 1

Schematic representation of CbMan5B/Cel44A, CbMan5B/Cel44A-WT, CbMan5B/Cel44A-TM1, CbMan5B/Cel44A-TM2, CbMan5B/Cel44A-TM3, and CbMan5B/Cel44A-TM4 of C. bescii. The putative functional domains were assigned by using the Pfam search tool (http://pfam.sanger.ac.uk/search/sequence). GH5, glycoside hydrolase family 5 catalytic module; GH44, glycoside hydrolase family 44 catalytic module; CBM3, carbohydrate-binding module family 3.

Fig 2

(A) Activities of CbMan5B/Cel44A-WT, CbMan5B/Cel44A-TM1, and CbMan5B/Cel44A-TM2 against polysaccharide substrates. CbMan5B/Cel44A-WT, CbMan5B/Cel44A-TM1, and CbMan5B/Cel44A-TM2 (0.5 μM) were incubated with each substrate (final concentration, 5 mg/ml) for 16 h at 70°C. The concentration of reducing ends was measured using the pHBAH assay. (B) Time course of hydrolysis of Avicel by CbMan5B/Cel44A-TM2. Avicel (10 mg/ml) was incubated with CbMan5B/Cel44A-TM2 (2 μM). At different time points, reaction mixtures were sampled, quenched, and diluted 10-fold in water and subjected to HPAEC-PAD analysis.

Fig 3

Optimal pH, optimal temperature, and thermostability assays of CbMan5B/Cel44A-TM2. (A) Optimal pH determination of CbMan5B/Cel44A-TM2. The CbMan5B/Cel44A-TM2 enzyme (2 μM) was incubated with 2.5 mg/ml phosphoric acid-swollen cellulose (PASC) in buffers differing in pH at 70°C. The released reducing ends were measured by the pHBAH assay. (B) Optimal temperature determination of CbMan5B/Cel44A-TM2. The CbMan5B/Cel44A-TM2 enzyme (2 μM) was incubated for 20 min with 2.5 mg/ml PASC in a citrate-HCl buffer (pH 5.0) at different temperatures ranging from 55 to 95°C, and the rates of end product release were determined. (C) Thermostability assay of CbMan5B/Cel44ATM2. The CbMan5B/Cel44ATM2 enzyme was incubated at 70°C, 75°C, 80°C, and 85°C. At different times, samples of the heated enzymes were taken and incubated with 2.5 mg/ml PASC dissolved in citrate-HCl buffer (pH 5.0) and reacted for 30 min at 70°C. After heat inactivation, the released reducing ends were measured as the residual activity using the pHBAH method.

Fig 4

Hydrolysis of cello-oligosaccharides (A), manno-oligosaccharides (B), and phosphoric acid-swollen cellulose (C) by CbMan5B/Cel44A-WT, CbMan5B/Cel44A-TM1, and CbMan5B/Cel44A-TM2. (A) Thin-layer chromatography (TLC) analysis of the reaction end products of CbMan5B/Cel44A-WT, TM1, and TM2 with glucose and cello-oligosaccharides. The reactions were carried out by incubating a 0.5 μM concentration of each protein with 5 mg/ml glucose or cello-oligosaccharide at 70°C for 16 h. At the end of the reaction, 1 μl of each reaction mixture was analyzed for end product release by TLC. G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose; G6, cellohexaose. (B) TLC analysis of the reaction products of CbMan5B/Cel44A-WT, CbMan5B/Cel44A-TM1, and CbMan5B/Cel44A-TM2 with mannose and manno-oligosaccharides. The reactions were carried out as described for panel A. The standards are as follows: M1, mannose; M2, mannobiose; M3, mannotriose; M4, mannotetraose; M5, mannopentaose; M6, mannohexaose. (C) Hydrolysis of PASC. The analyses were as described for panel A, except that the substrate was PASC at 10 mg/ml. At the end of the reaction, samples were analyzed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The standards for end product identification were G1 to G6.

Fig 5

Time course of hydrolysis of cellopentaose and PASC by CbMan5B/Cel44A-TM2. (A) Time course of hydrolysis of cellopentaose by CbMan5B/Cel44A-TM2. Cellopentaose (5 mg/ml) was incubated with 0.5 μM CbMan5B/Cel44A-TM2. At different time points, 200 μl of reaction mixture was sampled, quenched, diluted 50-fold in water, and subjected to HPAEC-PAD analysis. (B) The time course of hydrolysis of PASC by CbMan5B/Cel44A-TM2. PASC (5 mg/ml) was incubated with 1 μM CbMan5B/Cel44A-TM2, and at different time points, 200 μl of reaction mixture was sampled and the enzyme heat inactivated. Samples were then diluted 80-fold in water and subjected to HPAEC-PAD. The standards were glucose (G1), cellobiose (G2), cellotriose (G3), cellotetraose (G4), cellopentaose (G5), and cellohexaose (G6). (C) Based on the HPAEC-PAD profiles of the time course of hydrolysis of cellopentaose and PASC, the main cleavage pattern of CbMan5B/Cel44A-TM2 was proposed to be −4 to +n.

Fig 6

Synergistic effects of CbMan5B/Cel44A-TM2 with the endoglucanases CbCel9B/Man5A-TM1, CbMan5C/Cel5A-TM2, and CbCel5B-TM1 from C. bescii. (A) Schematic representation of CbMan5B/Cel44A-TM2, CbCel9B/Man5A-TM1, CbMan5C/Cel5A-TM2, and CbCel5B-TM1. (B) PASC (5 mg/ml) was incubated with individual enzymes (1 μM) or a combination of enzymes (1 μM each) in a citrate buffer (pH 5.5) at 70°C for 16 h. The end products were analyzed by HPAEC-PAD with G1 to G6 as standards. In panel B, values above the bars indicate degree of synergy (DOS). n.d., no synergism detected.

Fig 7

Time course hydrolysis of cellopentaose and PASC by CbCel9B/Man5A-TM1, CbMan5C/Cel5A-TM2, and CbCel5B-TM1. (A-i, A-ii, and A-iii) Time course of hydrolysis of cellopentaose by CbCel9B/Man5A-TM1, CbMan5C/Cel5A-TM2, and CbCel5B-TM1. Cellopentaose (5 mg/ml) was incubated with 0.5 μM each enzyme. Two hundred microliters of samples was removed, heat inactivated, diluted 50-fold, and analyzed by HPAEC-PAD. (B-i, B-ii, and B-iii) Time course of hydrolysis of PASC by CbCel9B/Man5A-TM1, CbMan5C/Cel5A-TM2, and CbCel5B-TM1. PASC (5 mg/ml) was incubated with each enzyme at 1 μM. Two hundred microliters of samples was removed, heat inactivated, diluted 80-fold, and analyzed by HPAEC-PAD. The standards were G1 to G6.