Bacillus licheniformistrehalose-6-phosphate hydrolase structures suggest keys to substrate specificity

2016 ◽  
Vol 72 (1) ◽  
pp. 59-70 ◽  
Author(s):  
Min-Guan Lin ◽  
Meng-Chun Chi ◽  
Vankadari Naveen ◽  
Yi-Ching Li ◽  
Long-Liu Lin ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Bacillus Licheniformis ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Positive Charge ◽  
Glycoside Hydrolase Family ◽  
Erwinia Rhapontici ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13

Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures ofBacillus licheniformisTreA (BlTreA) and its R201Q mutant complexed withp-nitrophenyl-α-D-glucopyranoside (R201Q–pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure ofBlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site ofBlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions ofBacillus cereusoligo-1,6-glucosidase (BcOgl) andErwinia rhaponticiisomaltulose synthase (NX-5), the surface potential of theBlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity ofBlTreA. Strikingly, the281HHLK284motif and Lys292 play critical roles in substrate discrimination byBlTreA.

scholarly journals Evolutionary variance analysis of the Glycoside Hydrolase Family 13: Structural evidence in classification and evolution

2017 ◽  
Author(s):  
Jose Sergio Hleap ◽  
Christian Blouin
Keyword(s):  
Glycoside Hydrolase ◽  
Structural Information ◽  
Industrial Applications ◽  
Glycoside Hydrolase Family ◽  
Sequence Information ◽  
Novel Method ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13 ◽  
Structural Shifts

AbstractGlycoside Hydrolase Family 13 (GH13) structures are responsible for the hydrolysis of starch into smaller carbohydrates. They important in industrial applications and evolutionary studies. This family has been thoroughly documented in the the Carbohydrate-Active enZYmes Database (CAZY), and divided into subfamilies based mainly in sequence information. Here we give structural evidence into GH13 classification and evolution using structural information. Here we proposed a novel method that is sensitive enough to identify miss-classifications, or to provide evidence for further partition that can be of interests to bio-engineers and evolutionary biologists. We also introduced a method to explore the relative importance of residues with respect to the overall deformation that it causes to the overall structure in an evolutionary time scale. We found that the GH13 family can be classified into three main structural groups. There is a hierarchical structure within these clusters that can be use to inform other classification schemes. We also found that by using structural information, subtle structural shifts can be identified and that can be missed in sequence/phylogeny-only based classifications. When each structural group is explored, we found that identifying the most structurally variable sites can lead to identification of functionally (both catalytically and structurally) important residues.

Relationship between the induced-fit loop and the activity of Klebsiella pneumoniae pullulanase

2019 ◽  
Vol 75 (9) ◽  
pp. 792-803
Author(s):  
Naoki Saka ◽  
Dominggus Malle ◽  
Hiroyuki Iwamoto ◽  
Nobuyuki Takahashi ◽  
Kimihiko Mizutani ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Binding Affinity ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Side Chain ◽  
Glycoside Hydrolase Family ◽  
Induced Fit ◽  
Structure And Activity ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13

Klebsiella pneumoniae pullulanase (KPP) belongs to glycoside hydrolase family 13 subfamily 13 (GH13_13) and is the only enzyme that is reported to perform an induced-fit motion of the active-site loop (residues 706–710). Comparison of pullulanase structures indicated that only KPP has Leu680 present behind the loop, in contrast to the glycine found in other GH13_13 members. Analysis of the structure and activity of recombinant pullulanase from K. pneumoniae ATCC 9621 (rKPP) and its mutant (rKPP-G680L) indicated that the side chain of residue 680 is important for the induced-fit motion of the loop 706–710 and alters the binding affinity of the substrate.

scholarly journals Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yuya Kuritani ◽  
Kohei Sato ◽  
Hideo Dohra ◽  
Seiichiro Umemura ◽  
Motomitsu Kitaoka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Metabolic Pathway ◽  
Glycoside Hydrolase ◽  
Gene Locus ◽  
Glycoside Hydrolase Family ◽  
Sequential Reaction ◽  
Layer Chromatography ◽  
Hydrolysis Of ◽  
Hydrolase Family

AbstractLevoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-d-glucose by LgdB2; next, LgdC reduces 3-keto-d-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

scholarly journals Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

2015 ◽  
Vol 290 (19) ◽  
pp. 11819-11832 ◽  
Author(s):  
Christopher M. Bianchetti ◽  
Taichi E. Takasuka ◽  
Sam Deutsch ◽  
Hannah S. Udell ◽  
Eric J. Yik ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Hydrolase Family

scholarly journals Epoxyalkyl glycosides of d-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10

2000 ◽  
Vol 347 (3) ◽  
pp. 865-873 ◽  
Author(s):  
Patricia NTARIMA ◽  
Wim NERINCKX ◽  
Klaus KLARSKOV ◽  
Bart DEVREESE ◽  
Mahalingeshwara K. BHAT ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolases ◽  
Thermomyces Lanuginosus ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
X Ray ◽  
Order Of Magnitude ◽  
Site Markers ◽  
Hydrolase Family

A series of Ω-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families 10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant to electrophilic attack of active-site carboxyl residues, glycoside hydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. The apparent inactivation and association constants (ki, 1/Ki) are one order of magnitude higher for the xylobiose and xylotriose derivatives. The effects of the aglycone chain length can clearly be described. Xylobiose and n-alkyl β-D-xylopyranosides are competitive ligands and provide protection against inactivation. MS measurements showed 1:1 stoichiometries in most labelling experiments. Electrospray ionization MS/MS analysis revealed the nucleophile Glu86 as the modified residue in the T. lanuginosus xylanase when 2,3-epoxypropyl β-D-xylopyranoside was used, whereas the acid/base catalyst Glu178 was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu86 and Glu177 in T. reesei Xyn II are similarly modified, confirming earlier X-ray crystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996) Biochemistry 35, 9617-9624]. The inability of the Ω-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10 enzymes is discussed in terms of different ligand-subsite interactions.

scholarly journals The response to selection in Glycoside Hydrolase Family 13 structures: A comparative quantitative genetics approach

2017 ◽  
Author(s):  
Jose Sergio Hleap ◽  
Christian Blouin
Keyword(s):  
Glycoside Hydrolase ◽  
Fitness Landscape ◽  
Protein Structures ◽  
Purifying Selection ◽  
Industrial Applications ◽  
Glycoside Hydrolase Family ◽  
Response To Selection ◽  
Effect Change ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13

AbstractThe Glycoside Hydrolase Family 13 (GH13) is both evolutionary diverse and relevant to many industrial applications. Its members perform the hydrolysis of starch into smaller carbohydrates. Members of the family have been bioengineered to improve catalytic function under industrial environments. We introduce a framework to analyze the response to selection of GH13 protein structures given some phylogenetic and simulated dynamic information. We found that the TIM-barrel is not selectable since it is under purifying selection. We also show a method to rank important residues with higher inferred response to selection. These residues can be altered to effect change in properties. In this work, we define fitness as inferred thermodynamic stability. We show that under the developed framework, residues 112Y, 122K, 124D, 125W, and 126P are good candidates to increase the stability of the truncated protein 4E2O. Overall, this paper demonstrate the feasibility of a framework for the analysis of protein structures for any other fitness landscape.

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