The chemical mechanism of a retaining β-mannosidase from Cellulomonas fimi has been characterized through steady-state kinetic analyses with a range of substrates, coupled with chemical rescue ...studies on both the wild-type enzyme and mutants in which active site carboxyl groups have been replaced. Studies with a series of aryl β-mannosides of vastly different reactivities (pK a lg = 4−10) allowed kinetic isolation of the glycosylation and deglycosylation steps. Substrate inhibition was observed for all but the least reactive of these substrates. Brønsted analysis of k cat revealed a downward breaking plot (βlg = −0.54 ± 0.05) that is consistent with a change in rate-determining step (glycosylation to deglycosylation), and this was confirmed by partitioning studies with ethylene glycol. The pH dependence of k cat/K m follows an apparent single ionization of a group of pK a = 7.65 that must be protonated for catalysis. The tentative assignment of E429 as the acid−base catalyst of Man2A on the basis of sequence alignments with other family 2 glycosidases was confirmed by the increased turnover rate observed for the mutant E429A in the presence of azide and fluoride, leading to the production of β-mannosyl azide and β-mannosyl fluoride, respectively. A pH-dependent chemical rescue of E429A activity is also observed with citrate. Substantial oxocarbenium ion character at the transition state was demonstrated by the α-deuterium kinetic isotope effect for Man2A E429A of α-D( V ) = 1.12 ± 0.01. Surprisingly, this isotope effect was substantially greater in the presence of azide (α-D( V ) = 1.166 ± 0.009). Likely involvement of acid/base catalysis was revealed by the pH dependence of k cat for Man2A E429A, which follows a bell-shaped profile described by pK a values of 6.1 and 8.4, substantially different from that of the wild-type enzyme. The glycosidic bond cleaving activity of Man2A E519A and E519S nucleophile mutants is restored with azide and fluoride and appears to correlate with the corresponding “glycosynthase” activities. The contribution of the substrate 2-hydroxyl to stabilization of the Man2A glycosylation transition state (ΔΔG ⧧ = 5.1 kcal mol-1) was probed using a 2-deoxymannose substrate. This value, surprisingly, is comparable to that found from equivalent studies with β-glucosidases despite the geometric differences at C-2 and the importance of hydrogen bonding at that position. Modes of stabilizing the mannosidase transition state are discussed.
The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and beta-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, ...comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian beta-mannosidases.
Endo-1,4-beta-mannanases (beta-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a ...major part of the plant cell-wall. The beta-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 beta-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by H-1-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10(-5) s(-1) at 5 degrees C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many 'retaining' enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the -3 subsite had a greater impact on the apparent k(cat)/K-m ratio (the catalytic efficiency) than the +2 subsite. The beta-anomer of mannotriose was hydrolysed 1000-times more efficiently than the alpha-anomer indicating selectivity for the beta- over the alpha-anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The Gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular beta-N -acetylglucosaminidases, a family 20 beta-N-acetylhexosaminidase (Hex20), and a novel family ...3-beta-N-acetylglucosaminidase/beta-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specidegrees C were 0.066 s-1 x mm-1 and 0.076 s-1 x mm-1 for 4'-nitrophenyl beta-N -acetyl-d-glucosaminide and 4'-nitrophenyl beta-d-glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between beta-N -acetylglucosaminidases and beta-glucosidases of family 3. Reaction by a double-displacement mechanism was confirmed for Nag3 through the identification of a glycosyl-enzyme species trapped with the slow substrate 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-d-glucopyranoside. Hex20 requires the acetamido group at C2 of the substrate, being unable to cleave beta-glucosides, since its mechanism involves an oxazolinium ion intermediate. However, it is broad in its specificity for thed-glucosyl/d-galactosyl configuration of the glycone: K m and k cat values were 53 micro m and 482.3 s-1 for 4'-nitrophenyl beta-N -acetyl-d-glucosaminide and 66 micro m and 129.1 s-1 for 4'-nitrophenyl beta-N -acetyl-d-galactosaminide.PUBLICATION ABSTRACT
Enzymatic Synthesis of Carbon−Fluorine Bonds Zechel, David L; Reid, Stephen P; Nashiru, Oyekanmi ...
Journal of the American Chemical Society,
05/2001, Letnik:
123, Številka:
18
Journal Article
Endo-1,4-β-mannanases (β-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part ...of the plant cell-wall. The β-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 β-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by
1
H-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10
−5
s
−1
at 5°C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many 'retaining' enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the −3 subsite had a greater impact on the apparent k
cat
/K
m
ratio (the catalytic efficiency) than the +2 subsite. The β-anomer of mannotriose was hydrolysed 1000-times more efficiently than the α-anomer indicating selectivity for the β- over the α-anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Endo-1,4- -mannanases ( -mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part ...of the plant cell-wall. The -mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 -mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by 1H-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10−5s−1 at 5°C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many 'retaining' enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the −3 subsite had a greater impact on the apparent kcat/Km ratio (the catalytic efficiency) than the +2 subsite. The -anomer of mannotriose was hydrolysed 1000-times more efficiently than the -anomer indicating selectivity for the - over the -anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
A modular mannanase (Man26A) from the bacterium
Cellulomonas fimi contains a mannan-binding module (Man26Abm) that binds to soluble but not to insoluble mannans. Man26Abm does not bind to cellulose, ...chitin or xylan. The
K
d for binding of Man26Abm to locust bean gum (LBG) is ∼0.2 μM. Man26A is the first mannanase reported to contain a mannan-binding module.
In this study the mannan-degrading system of the Gram positive bacterium
Cellulomonas fimi was characterised. C. fimi can degrade different forms of mannan and can
use the degradation products as ...carbon and energy source. This study focuses on the
galactomannan-degrading system which was found to be composed of one secreted endo-
1,4- β -mannanase (Man26A), one intracellular l,4-β-mannosidase (Man2A) and one
intracellular 1,6-α-galactosidase. The genes encoding Man26A and Man2A have been
isolated and sequenced, and the enzyme activities were investigated.
The endo-l,4- β -mannanase (Man26A) has a multidomain structure and comprises a
family 26 catalytic domain, a mannan-binding domain (MBD), a S-layer homology domain
(SLH domain) and a domain of yet unknown function. Mannanase activity was detected on
the cell surface and in the culture supernatant. It is believed that the SLH domain mediates
transient binding of Man26A to the cell surface and the MBD mediates binding to the
substrate. Strong binding of the MBD to soluble mannan was detected and its potential as
an affinity tag for protein purification in aqueous 2-phase systems was tested.
The 1,4- β -mannosidase (Man2A), cleaves β -1,4 mannosidic linkages with net retention
of the anomeric configuration. Man2A was transformed into the glycosynthase Man2A
E519A by mutating the catalytic nucleophile, E519 to alanine. Glycosynthases are retaining
glycosidases without hydrolytic but with synthetic activity. Using α-mannosyl fluoride as
donor and p-nitrophenyl sugars as acceptors, the glycosynthase Man2A E519A catalyzed
the synthesis of β -1,4 and β -1,3 mannosidic linkages.
In this study the biology of mannan-degradation by C. fimi was investigated and the
biotechnological potential of its components was explored.
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