Substrate specificity and subsite mobility in <i>T. aurantiacus</i> xylanase 10A

Leggio, Leila Lo; Kalogiannis, Stavros; Eckert, Kelvin; Teixeira, Susana; Bhat, M. K.; Andrei, Carmen; Pickersgill, Richard W.; Larsen, Sine

doi:10.1016/s0014-5793(01)03177-5

Cited by 53 publications

(51 citation statements)

References 40 publications

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“…For the TaXyn10A structure it was concluded that if the À1 subsite xylose were in the b-configuration, a steric clash would likely induce ring strain, with implications for catalysis. The authors further suggested that the a-configuration as observed in TaXyn10A and now PbXynA1CD, is due to the enzyme selecting the anomeric configuration that most resembles the covalent intermediate step of the double-displacement mechanism (Lo Leggio et al, 2001). Our structure analysis of PbXynA1CD supports these earlier findings and their conclusions.…”

Section: Coordination Of Megx 3 In the Glycone Subsitessupporting

confidence: 87%

“…In the case of Cellvibrio mixtus Xyn10B (CmXyn10B) (PDB code: 1UQZ) (Pell et al, 2004b), a catalytic nucleophile mutation to a serine prevents meaningful comparison. The glycone region of native Thermoascus aurantiacus xylanase A (TaXyn10A) (PDB code: 1GOR) contains X 2 with the À1 subsite xylose in an a-anomeric conformation and similar coordination position as observed for MeGX 3 -bound PbXynA1CD (Lo Leggio et al, 2001). For the TaXyn10A structure it was concluded that if the À1 subsite xylose were in the b-configuration, a steric clash would likely induce ring strain, with implications for catalysis.…”

Section: Coordination Of Megx 3 In the Glycone Subsitesmentioning

confidence: 95%

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Novel structural features of xylanase A1 from Paenibacillus sp. JDR-2

John

Preston

Pozharski

2012

Journal of Structural Biology

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a b s t r a c tThe Gram-positive bacterium Paenibacillus sp. JDR-2 (PbJDR2) has been shown to have novel properties in the utilization of the abundant but chemically complex hemicellulosic sugar glucuronoxylan. Xylanase A1 of PbJDR2 (PbXynA1) has been implicated in an efficient process in which extracellular depolymerization of this polysaccharide is coupled to assimilation and intracellular metabolism. PbXynA1is a 154 kDa cell wall anchored multimodular glycosyl hydrolase family 10 (GH10) xylanase. In this work, the 38 kDa catalytic module of PbXynA1 has been structurally characterized revealing several new features not previously observed in structures of GH10 xylanases. These features are thought to facilitate hydrolysis of highly substituted, chemically complex xylans that may be the form found in close proximity to the cell wall of PbJDR2, an organism shown to have a preference for growth on polymeric glucuronoxylan.Published by Elsevier Inc.

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Section: Coordination Of Megx 3 In the Glycone Subsitessupporting

confidence: 87%

Section: Coordination Of Megx 3 In the Glycone Subsitesmentioning

confidence: 95%

Novel structural features of xylanase A1 from Paenibacillus sp. JDR-2

John

Preston

Pozharski

2012

Journal of Structural Biology

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“…The active site search was carried out in the xylan binding pocket of the xylanase from Thermoascus aurantiacus (TAX) (19); multiple active site configurations were identified. One of the best scoring active sites included the wild-type glutamate at position 237, which is predicted to serve as a general base for the reaction and adopts a conformation similar to that seen in the crystal structure of wild-type TAX.…”

Section: Resultsmentioning

confidence: 99%

Iterative approach to computational enzyme design

Privett

Kiss

Lee

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

301

357

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A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes.computational protein design | de novo enzyme design | proton transfer T he high efficiency, chemoselectivity, regio-and stereospecificity, and biodegradability of enzymes make them extremely attractive catalysts. However, the finite repertoire of naturally occurring enzymes limits their applicability to broad problems in biotechnology. A general method for the computational design of enzymes that can efficiently catalyze arbitrary chemical reactions would allow the benefits of enzymatic catalysis to be applied to chemical transformations of interest that are currently inaccessible via natural enzymes. Bolon and Mayo provided important early evidence that such an approach is feasible (1), which motivated significant progress toward this goal in recent years. Using quantum mechanics-based active site design and the Rosetta software suite, Baker, Houk, and coworkers designed enzymes for three chemically unrelated nonnatural reactions in a variety of catalytically inert scaffolds (2-4).In early incarnations of computational protein design, a strategy for methods development was put forth in terms of the so-called "protein design cycle" in which experimental evaluation of an initial design is used to inform adjustments to the design process for subsequent rounds of design (5, 6). Ideally, these steps would be continued iteratively until the protein sequences predicted by the algorithm exhibit the desired char...

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“…This hydrogen atom fits neatly into a pocket comprising W316 and W324 ( Fig. 5; 1R87 numbering), which is part of a highly conserved tryptophan cage [42,46]. Three contacts under 3 A were identified between the 5b equatorial hydrogen at subsite À1 and XT6 residue W324.…”

Section: X2: Binding Of Wild-type Versus E159q Xt6mentioning

confidence: 98%

Identifying critical unrecognized sugar–protein interactions in GH10 xylanases from Geobacillus stearothermophilus using STD NMR

et al. 2013

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1H solution NMR spectroscopy is used synergistically with 3D crystallographic structures to map experimentally significant hydrophobic interactions upon substrate binding in solution under thermodynamic equilibrium. Using saturation transfer difference spectroscopy (STD NMR), a comparison is made between wild‐type xylanase XT6 and its acid/base catalytic mutant E159Q – a non‐active, single‐heteroatom alteration that has been previously utilized to measure binding thermodynamics across a series of xylooligosaccharide–xylanase complexes [Zolotnitsky et al. (2004) Proc Natl Acad Sci USA 101, 11275–11280). In this study, performing STD NMR of one substrate screens binding interactions to two proteins, avoiding many disadvantages inherent to the technique and clearly revealing subtle changes in binding induced upon mutation of the catalytic Glu. To visualize and compare the binding epitopes of xylobiose–xylanase complexes, a ‘SASSY’ plot (saturation difference transfer spectroscopy) is used. Two extraordinarily strong, but previously unrecognized, non‐covalent interactions with H2–5 of xylobiose were observed in the wild‐type enzyme but not in the E159Q mutant. Based on the crystal structure, these interactions were assigned to tryptophan residues at the −1 subsite. The mutant selectively binds only the β–xylobiose anomer. The 1H solution NMR spectrum of a xylotriose–E159Q complex displays non‐uniform broadening of the NMR signals. Differential broadening provides a unique subsite assignment tool based on structural knowledge of face‐to‐face stacking with a conserved tyrosine residue at the +1 subsite. The results obtained herein by substrate‐observed NMR spectroscopy are discussed further in terms of methodological contributions and mechanistic understanding of substrate‐binding adjustments upon a charge change in the E159Q construct.

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Substrate specificity and subsite mobility in T. aurantiacus xylanase 10A

Cited by 53 publications

References 40 publications

Novel structural features of xylanase A1 from Paenibacillus sp. JDR-2

Novel structural features of xylanase A1 from Paenibacillus sp. JDR-2

Iterative approach to computational enzyme design

Identifying critical unrecognized sugar–protein interactions in GH10 xylanases from Geobacillus stearothermophilus using STD NMR

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