Crystal structure of AmyA lacks acidic surface and provide insights into protein stability at poly‐extreme condition

Sivakumar, Neelamegam; Li, Nan; Tang, Julian W.; Patel, Bharat K. C.; Swaminathan, Kunchithapadam

doi:10.1016/j.febslet.2006.04.017

Cited by 67 publications

(51 citation statements)

References 28 publications

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“…Halothermothrix orenii a-amylase (GH13_36) [22][23][24][25][26][27]. Two orientation patterns of the Trp side-chain are observed in GH13 enzymes with known structures.…”

Section: Structural Insights Into Catalytic Reaction Involving Reoriementioning

confidence: 99%

Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate‐binding site in GH13 dextran glucosidase

et al. 2015

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a b s t r a c tStreptococcus mutans dextran glucosidase (SmDG) belongs to glycoside hydrolase family 13, and catalyzes both the hydrolysis of substrates such as isomaltooligosaccharides and subsequent transglucosylation to form a-(1 ? 6)-glucosidic linkage at the substrate non-reducing ends. Here, we report the 2.4 Å resolution crystal structure of glucosyl-enzyme intermediate of SmDG. In the obtained structure, the Trp238 side-chain that constitutes the substrate-binding site turned away from the active pocket, concurrently with conformational changes of the nucleophile and the acid/base residues. Different conformations of Trp238 in each reaction stage indicated its flexibility. Considering the results of kinetic analyses, such flexibility may reflect a requirement for the reaction mechanism of SmDG.

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“…Halothermothrix orenii a-amylase (GH13_36) [22][23][24][25][26][27]. Two orientation patterns of the Trp side-chain are observed in GH13 enzymes with known structures.…”

Section: Structural Insights Into Catalytic Reaction Involving Reoriementioning

confidence: 99%

Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate‐binding site in GH13 dextran glucosidase

et al. 2015

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“…Halophilic and halo-tolerant microorganisms have been the resource of halophilic enzymes that can function in the presence of high salt concentrations or under low-water activity conditions [5,16,17]. Several halophilic and halo-tolerant a-amylases, b-amylase, branching enzyme and neopullulanase were isolated from extremely and moderately halophilic microorganisms [12,19,21,33,35] and polyextremophilic microorganisms, e.g., thermophilic halophile [23,27]. However, the studies of starch-binding modules from halophilic enzymes have been scarce [27,34].…”

Section: Discussionmentioning

confidence: 99%

Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic Kocuria varians Alpha-Amylase

et al. 2012

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Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity from Escherichia coli. The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure. This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw starch granules and amylose resin with 70-80% efficiency compared with binding of equimolar His-SBD12. Although the thermal unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different: His-SBD12 refolded slowly (T(1/2) = ~84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T(1/2) = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5-6 °C increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding were insignificant.

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“…Catalytic Mechanism-The substrate recognition scheme and binding sites for several amylase family enzymes, like PalI (33), AmyA from Halothermothrix orenii (38), ␣-amylase (39), CGTase (40), TAKA-amylase with substrate analogs (41), amylosucrase with D-glucose and mutated amylosucrase with sucrose (42,43), have been identified. A substrate binding model has been proposed for EC⌬112GlgB (21).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme

Pal

Kumar

Sharma

et al. 2010

Journal of Biological Chemistry

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The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an ␣-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 134 bond and making a new 136 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-Å resolution. MtbGlgBWT contains four domains: N1 ␤-sandwich, N2 ␤-sandwich, a central (␤/␣) 8 domain that houses the catalytic site, and a C-terminal ␤-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb⌬108GlgB protein. The N1 ␤-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 ␤-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb⌬108GlgB. We propose a twostep reaction mechanism, for the amylase activity (134 bond breakage) and isomerization (136 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb⌬108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC⌬112GlgB).Tuberculosis is still a major killer infectious disease, at least in developing countries. Mycobacterium tuberculosis (Mtb), 7 the causative bacterial agent of tuberculosis, survives for a long period of time intracellularly and causes latent tuberculosis. If the organism is physiologically inactive for a long period of time, its storage sugars become very important for its survival. Therefore, understanding the nature of the enzymes that are involved in the metabolism of these storage sugars is very important. Glycogen is one of the most important storage sugars in the living world and provides nutrition to the host. Furthermore, the cell envelop of Mtb has a very important role during host-pathogen interactions. The outermost layer of the cell envelop of Mtb consists of a loosely bound structure, known as the capsule (1-3). It has been demonstrated that the major components of the capsular material are carbohydrates and proteins with a very small amount of lipids (4 -6). The major carbohydrate constituent (ϳ80%) of the Mtb capsule is a high molecular mass (Ͼ100,000 Da) ␣-glucan, which is composed of a (34-␣-D-Glc-13) core and branched at position 6, every 5 or 6 residues, by (34-␣-D-Glc-13) oligoglucosides (4,7,8). ␣-Glucan mediates non-opsonic binding of Mtb to CR3 (complement receptor3) (9) and is instrumental in blocking CD1 expression in Mtb (10). Stokes et al. (11) have shown that the capsular material of Mtb also displays antiphagocytic properties with certain types of macrophages.Glycogen synthesis is an endergonic process. Glycogen, composed of branched polymer chains ...

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Crystal structure of AmyA lacks acidic surface and provide insights into protein stability at poly‐extreme condition

Cited by 67 publications

References 28 publications

Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate‐binding site in GH13 dextran glucosidase

Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate‐binding site in GH13 dextran glucosidase

Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic Kocuria varians Alpha-Amylase

Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme

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