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Prot Cell    2011, Vol. 2 Issue (10) : 827-836    https://doi.org/10.1007/s13238-011-1105-3      PMID: 22058037
RESEARCH ARTICLE
Structural insight into substrate specificity of human intestinal maltase-glucoamylase
Limei Ren1,2, Xiaohong Qin1,3, Xiaofang Cao1,2, Lele Wang1,3, Fang Bai2, Gang Bai1,2(), Yuequan Shen1,3()
1. State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China; 2. College of Pharmacy, Nankai University, Tianjin 300071, China; 3. College of Life Sciences, Nankai University, Tianjin 300071, China
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Abstract

Human maltase-glucoamylase (MGAM) hydrolyzes linear alpha-1,4-linked oligosaccharide substrates, playing a crucial role in the production of glucose in the human lumen and acting as an efficient drug target for type 2 diabetes and obesity. The amino- and carboxyl-terminal portions of MGAM (MGAM-N and MGAM-C) carry out the same catalytic reaction but have different substrate specificities. In this study, we report crystal structures of MGAM-C alone at a resolution of 3.1 ?, and in complex with its inhibitor acarbose at a resolution of 2.9 ?. Structural studies, combined with biochemical analysis, revealed that a segment of 21 amino acids in the active site of MGAM-C forms additional sugar subsites (+2 and+3 subsites), accounting for the preference for longer substrates of MAGM-C compared with that of MGAM-N. Moreover, we discovered that a single mutation of Trp1251 to tyrosine in MGAM-C imparts a novel catalytic ability to digest branched alpha-1,6-linked oligosaccharides. These results provide important information for understanding the substrate specificity of alpha-glucosidases during the process of terminal starch digestion, and for designing more efficient drugs to control type 2 diabetes or obesity.
Keywords MGAM C-terminal domain      inhibitor      crystal structure      acarbose      type 2 diabetes     
Corresponding Author(s): Bai Gang,Email:gangbai@nankai.edu.cn (G. Bai); yshen@nankai.edu.cn; Shen Yuequan,Email:yuequan74@yahoo.com (Y. Shen)   
Issue Date: 01 October 2011
 Cite this article:   
Limei Ren,Lele Wang,Fang Bai, et al. Structural insight into substrate specificity of human intestinal maltase-glucoamylase[J]. Prot Cell, 2011, 2(10): 827-836.
 URL:  
https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1105-3
https://academic.hep.com.cn/pac/EN/Y2011/V2/I10/827
Fig.1  Structure of the MGAM-C/acarbose complex.
(A) Schematic representation of human MGAM domains with amino-acid boundaries; (B) Schematic drawing of the structure of acarbose; (C) Ribbon diagram of the MGAM-C/acarbose complex. Individual domains are colored as follows: trefoil Type-P domain (blue), N-terminal domain (yellow), catalytic (β/α)8 domain (red), catalytic domain Insert 1 (orange), catalytic domain Insert 2 (pink), proximal C-terminal domain (ProxC) (green), and distal C-terminal domain (DistC) (purple). The bound inhibitor acarbose is shown as a stick model and colored cyan. N, N terminal; C, C terminal.
Fig.1  Structure of the MGAM-C/acarbose complex.
(A) Schematic representation of human MGAM domains with amino-acid boundaries; (B) Schematic drawing of the structure of acarbose; (C) Ribbon diagram of the MGAM-C/acarbose complex. Individual domains are colored as follows: trefoil Type-P domain (blue), N-terminal domain (yellow), catalytic (β/α)8 domain (red), catalytic domain Insert 1 (orange), catalytic domain Insert 2 (pink), proximal C-terminal domain (ProxC) (green), and distal C-terminal domain (DistC) (purple). The bound inhibitor acarbose is shown as a stick model and colored cyan. N, N terminal; C, C terminal.
Fig.2  Interaction of MGAM-C with acarbose.
(A) Stereo view of the 2Fo-Fc electron density map in the active site of the MGAM-C/acarbose structure contoured at the 2.0σ level and shown in blue. Acarbose is represented as thick cyan sticks, and the active-site residues are represented as thin green sticks. (B) The diagrammatic representations of the hydrogen bonds (dashed lines) and hydrophobic interactions (dashed-lined semicircles) formed by MGAM-C with inhibitor acarbose. Sugar subsites (-1, +1, +2 and+3) are labeled accordingly.
Fig.2  Interaction of MGAM-C with acarbose.
(A) Stereo view of the 2Fo-Fc electron density map in the active site of the MGAM-C/acarbose structure contoured at the 2.0σ level and shown in blue. Acarbose is represented as thick cyan sticks, and the active-site residues are represented as thin green sticks. (B) The diagrammatic representations of the hydrogen bonds (dashed lines) and hydrophobic interactions (dashed-lined semicircles) formed by MGAM-C with inhibitor acarbose. Sugar subsites (-1, +1, +2 and+3) are labeled accordingly.
SubstrateKm (mmol/L)
MGAMaMGAM-NMGAM-CMGAM-C-deltaS
Maltose2.16.40±0.585.67±0.265.91±0.72
Maltotriose1.14.44±0.570.91±0.111.89±0.37
Maltotetraose0.43.39±0.310.96±0.064.73±0.36
Maltopentaose0.49.14±0.240.61±0.068.24±1.25
Maltohexaose0.79.76±0.211.05±0.1012.33±0.91
Maltoheptaose113.12±2.602.27±0.3313.70±1.78
Tab.1  Kinetic parameters for wild-type and mutant MGAM
Fig.3  Substrate specificity of MGAM-C.
(A) Sequence alignment of crucial insertions in MGAM and SI enzymes. Highly conserved residues are colored white, and moderately conserved residues are colored red. (B) Superposition of MGAM-C/acarbose (light cyan), MGAM-N (light purple) and SI-N (pink) active sites. Acarbose is represented as thick cyan sticks, and the additional 21 amino acids in MGAM-C are colored red. The approximate locations of the –1 to+3 subsites in MGAM-C are labeled. (C) Surface representation of the MGAM-C (light cyan)/acarbose (cyan), MGAM-N (light purple)/acarbose (yellow) and SI-N (pink)/kotalanol (green) active sites, with non-structurally conserved residues displayed as green, purple and magenta sticks, respectively. The surface of the additional 21 amino acids in MGAM-C is colored red.
Fig.3  Substrate specificity of MGAM-C.
(A) Sequence alignment of crucial insertions in MGAM and SI enzymes. Highly conserved residues are colored white, and moderately conserved residues are colored red. (B) Superposition of MGAM-C/acarbose (light cyan), MGAM-N (light purple) and SI-N (pink) active sites. Acarbose is represented as thick cyan sticks, and the additional 21 amino acids in MGAM-C are colored red. The approximate locations of the –1 to+3 subsites in MGAM-C are labeled. (C) Surface representation of the MGAM-C (light cyan)/acarbose (cyan), MGAM-N (light purple)/acarbose (yellow) and SI-N (pink)/kotalanol (green) active sites, with non-structurally conserved residues displayed as green, purple and magenta sticks, respectively. The surface of the additional 21 amino acids in MGAM-C is colored red.
Maltose (α-1,4)Isomaltose (α-1,6)
SubstrateKm (mmol·L-1)Kcat (S-1)Kcat/Km (S-1/mmol·L-1)Km (mmol·L-1)Kcat (S-1)Kcat/Km (S-1/ mmol·L-1)
MGAM-N6.40±0.5848.62±8.937.5770.45±6.706.92±0.720.10
MGAM-N-Y299W8.81±0.4623.27±0.802.6423.46±4.454.60±0.210.20
MGAM-C5.67±0.2622.49±0.963.97N.D.N.D.N.D.
MGAM-C-Y1251W16.16±1.497.12±0.750.4449.95±1.030.39±0.020.01
Tab.2  Kinetic parameters for α-1,4 and α-1,6 substrate hydrolysis by MGAM mutants
Crystal nameMGAM-C aloneMGAM-C/acarbose
Space groupBoldItalic43212BoldItalic43212
Unit cell (?)a = b = 106.23, c = 517.56a = b = 105.50, c = 516.56
Wavelength (?)0.97940.9794
Resolution range (?)50-3.0 (3.1-3.0)50-2.9 (3.0-2.9)
No. of unique reflections50,13159,475
Redundancy8.6 (5.0)a13.3 (7.2)a
BoldItalicsym (%)b6.9 (38.3)a6.6 (43.7)a
BoldItalic/BoldItalic40.5 (2.2)a66.9 (2.2)a
Completeness (%) 99.4 (24.2)a99.3 (45.1)a
Refinement
Resolution range (?)50-3.150-2.9
Rcrystal (%)c23.221.9
Rfree (%)d28.828.4
RMSDbond (?)0.010.009
RMSDangle(°)1.311.27
Number of
protein atoms14,25214,352
ligand atoms088
solvent atoms2040
Residues in (%)
most favored 78.480.0
additional allowed20.118.4
generously allowed1.41.4
disallowed0.10.1
Average B factor (?2) of
protein58.988.4
ligand atoms-88.0
solvent atoms27.158.5
Tab.3  Data collection and refinement statistics
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