Theoretical Calculation of pKReveals an Important Role of Arg205 in the Activity and Stability of Streptomyces sp. N174 Chitosanase

Fukamizo, Tamo; Juffer, André H.; Vogel, Hans J.; Honda, Yoshiyuki; Tremblay, Hugo; Boucher, Isabelle; Neugebauer, Witold; Brzeziński, Ryszard

doi:10.1074/jbc.m002574200

Cited by 35 publications

(17 citation statements)

References 35 publications

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“…S7A). This is consistent with studies of chitosanases OU01 and N174 (30,31). Trp-273 in AxMan113A provided a hydrophobic sugar-binding platform with the ϩ1 sugar ring.…”

Section: Structural Analysis Of Gh Family 113 ␤-14-mannanasesupporting

confidence: 90%

Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 β-1,4-mannanase from Amphibacillus xylanus

You

Qin

Yan

et al. 2018

Journal of Biological Chemistry

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β-1,4-Mannanase degrades β-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) family 113 β-1,4-mannanase, from (ManA), has been structurally characterized, and no complex structure of enzyme-manno-oligosaccharides from this family has been reported. Here, crystal structures of a GH family 113 β-1,4-mannanase from (Man113A) and its complexes with mannobiose, mannotriose, mannopentaose, and mannahexaose were solved. Man113A had higher affinity for -1 and +1 mannoses, which explains why the enzyme can hydrolyze mannobiose. At least six subsites (-4 to +2) exist in the groove, but mannose units preferentially occupied subsites -4 to -1 because of steric hindrance formed by Lys-238 and Trp-239. Based on the structural information and bioinformatics, rational design was implemented to enhance hydrolysis activity. Enzyme activity ofMan113A mutants V139C, N237W, K238A, and W239Y was improved by 93.7, 63.4, 112.9, and 36.4%, respectively, compared with the WT. In addition, previously unreported surface-binding sites were observed. Site-directed mutagenesis studies and kinetic data indicated that key residues near the surface sites play important roles in substrate binding and recognition. These first GH family 113 β-1,4-mannanase-manno-oligosaccharide complex structures may be useful in further studying the catalytic mechanism of GH family 113 members, and provide novel insight into protein engineering of GHs to improve their hydrolysis activity.

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“…S7A). This is consistent with studies of chitosanases OU01 and N174 (30,31). Trp-273 in AxMan113A provided a hydrophobic sugar-binding platform with the ϩ1 sugar ring.…”

Section: Structural Analysis Of Gh Family 113 ␤-14-mannanasesupporting

confidence: 90%

Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 β-1,4-mannanase from Amphibacillus xylanus

You

Qin

Yan

et al. 2018

Journal of Biological Chemistry

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“…This Arg ( R DP motif) is completely conserved in clan GH-J while the Tyr ( Y ASK motif) is not [28]. Similar findings have been reported before in another enzyme clan [29]. Based on all these findings, an improved catalytic mechanism is proposed within the GH-J clan.…”

Section: Introductionsupporting

confidence: 83%

pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

et al. 2012

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Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst.

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“…Such residues can be involved in enzyme-substrate interaction, in structure stabilisation, but also in the creation of interaction networks essential for catalysis [6,11-13]. In CsnN174, examination of the microenvironment of the proton donor Glu22 revealed that this glutamate does not work independently but requires assistance from an interaction network involving three other residues (Arg205, Asp145 and Arg190) to achieve efficient catalysis [13]. Similar networks have also been observed in other members of the lysozyme superfamily, Bacillus circulans MH-K1 chitosanase (GH46), T4 lysozyme (GH24) and barley chitinase (GH19) [13].…”

Section: Introductionmentioning

confidence: 99%

“…In CsnN174, examination of the microenvironment of the proton donor Glu22 revealed that this glutamate does not work independently but requires assistance from an interaction network involving three other residues (Arg205, Asp145 and Arg190) to achieve efficient catalysis [13]. Similar networks have also been observed in other members of the lysozyme superfamily, Bacillus circulans MH-K1 chitosanase (GH46), T4 lysozyme (GH24) and barley chitinase (GH19) [13]. In order to verify if a similar kind of interaction is needed to sustain the catalytic potential of the general base residue, we examined the microenvironment of Asp40 of CsnN174 and found that an arginine (Arg42) is highly conserved among the GH46 family of chitosanases.…”

Section: Introductionmentioning

confidence: 99%

A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding

et al. 2013

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BackgroundStreptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42.ResultsMutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity.ConclusionsArginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan.

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Theoretical Calculation of pKReveals an Important Role of Arg205 in the Activity and Stability of Streptomyces sp. N174 Chitosanase

Cited by 35 publications

References 35 publications

Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 β-1,4-mannanase from Amphibacillus xylanus

Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 β-1,4-mannanase from Amphibacillus xylanus

pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding

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