Neopullulanase

Last updated
Neopullulanase
Neopulullanase ribbon rendering.png
View of Thermoactinomyces vulgaris neopulullanase showing dimeric structure. [1]
Identifiers
EC no. 3.2.1.135
CAS no. 119632-58-5
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Search
PMC articles
PubMed articles
NCBI proteins

Neopullulanase (EC 3.2.1.135, pullulanase II) is an enzyme of the alpha-amylase family with systematic name pullulan 4-D-glucanohydrolase (panose-forming). [2] This enzyme principally catalyses the following chemical reaction by cleaving pullulan's alpha-1,4-glucosidic bonds:

Contents

Hydrolysis of pullulan to panose (6-alpha-D-glucosylmaltose)

The breakdown of the alpha-1,4- and alpha-1,6-glucosidic bonds of intermediates produced in addition to panose generates further quantities of panose along with some maltose and glucose. [2]

Structure

Neopullulanase is a dimer of identical monomer subunits, each with four domains (N,A,B,C) that are highly conserved with other starch hydrolases, namely alpha-amylase, pullulanase, cyclomaltodextrin glucanotransferase, and 1,4-alpha-D-glucan branching enzyme (also known as glycogen branching enzyme). [3] Like these enzymes, each monomer contains an active site at the carboxyl-terminus within a TIM barrel (also known as an alpha/beta barrel), an alpha/beta protein fold structure consisting of eight parallel beta-strands connected by eight external alpha-helices. [1]

This conserved structural domain is estimated to occur in roughly 10% of all proteins and may evolutionarily link neopullulanase and the similar starch hydrolases to a much larger family of enzymes, though the domain's common ancestry is debated due to a lack of conclusive sequence homology. [4] In neopullulanase the barrel is located within domain A with its active site straddling domain A of one monomer with domain N of the other monomer. This results in narrower active site than the other alpha-amylase enzymes, which do not dimerize, and likely contributes to its ability of hydrolyzing both alpha-1,4- and alpha-1,6-glucosidic linkages. [5]

Mechanism

The hydrolysis of pullulan to panose is catalyzed by three amino acid residues within neopullulanase's active site that cleave a glycosidic bond: one glutamate and two aspartates. [6] A glycosidic oxygen is first protonated by the carboxyl group of a glutamate residue (TAA Glu-230) through generic acid catalysis. The C1 carbon of pullulan is then attacked by a nucleophilic aspartate residue (TAA Asp-206). The carboxylate group of a second aspartate residue (TAA Asp-297) deprotonates an adjacent water molecule to form a hydroxide ion which hydroxylates at the C1 carbon. Alternatively it is possible that this reaction is concerted with the departed glycosidic oxygen being protonated to cause the hydroxylation.

Mechanism of neopullulanase catalyzing hydrolysis of pullulan decomposition intermediate into panose and maltose. Neopullulanase mechanism.png
Mechanism of neopullulanase catalyzing hydrolysis of pullulan decomposition intermediate into panose and maltose.

The three residues responsible for neopullulanase catalysis are invariably present in enzymes of the alpha-amylase family. [6] Mutation of these residues in neopullulanase results in a complete loss of enzymatic activity.

While most alpha-amylase enzymes only cleave alpha-1,4-linkages in their substrates, neopullulanase additionally cleaves alpha-1,6-linkages. [6] In addition to the narrowness of the actives site resulting from the enzyme's dimeric structure, this additional functionality is thought to be facilitated by two histidine residues (TAA His-122 and TAA His-296) that interact with the glycan bond to be cleaved. As these histidines are present in the other alpha-amylase enzymes it is thought the functional difference arises from a difference in transition state stabilization energy contributions from the side chains of adjacent residues which vary from enzyme to enzyme.

This allows for neopullulanase's multistep breakdown of pullulan. The enzyme first selectively hydrolyzes alpha-1,4-glucosidic bonds on the nonreducing side of pullulan's alpha-1,6-glucosidic bonds, producing panose and panose-containing intermediates. These intermediates then have their alpha-1,4- and alpha-1,6-glucosidic bonds hydrolyzed to form additional panose along with smaller quantities of maltose and glucose.

Biological Function

Pullulan, which is produced from starch, is a polysaccharide polymer consisting of repeating maltotriose units. It provides a protective effect against cellular desiccation in low-moisture environments. [7]

The presence of neopullulanase allows cells to recycle unneeded or excess pullulan by breaking it down into panose, maltose, and glucose which can then be formed back into starch or consumed for energy production.

Industrial Relevance

While not currently employed in any industrial processes, a method of producing isomaltooligosaccharide syrup using Bacillus stearothermophilus neopullulase has been proposed, taking advantage of neopullulase's ability to catalyze hydrolysis of branched oligosaccharides' alpha-1-6-glucosidic linkages. [8] While primarily used as a source for dietary fiber, isomaltooligosaccharide syrup is also used as a low-calorie sweetener that can reduce the buildup of dental plaque when present in place of sucrose. [9]

This process is simpler than the currently prevalent industrial process which relies upon multiple steps featuring four enzymes (alpha-amylase, pullulanase, beta-amylase, and alpha-D-glucosidase) and only achieves a 40% yield of isomaltooligosaccharides from starch. When immobilized neopullulanase is immersed in a buffered starch solution and incubated, a solution of isomaltooligosaccharides results at slightly over 40% yield. To further raise the yield to approximately 60%, which is thought to be so low since neopullulanase hydrolyzes starch less efficiently than pullunan and other oligosaccharides, saccharifying alpha-amylase sourced from Bacillus subtilis may be added to the solution. [8]

See also

Related Research Articles

<span class="mw-page-title-main">Amylase</span> Class of enzymes

An amylase is an enzyme that catalyses the hydrolysis of starch into sugars. Amylase is present in the saliva of humans and some other mammals, where it begins the chemical process of digestion. Foods that contain large amounts of starch but little sugar, such as rice and potatoes, may acquire a slightly sweet taste as they are chewed because amylase degrades some of their starch into sugar. The pancreas and salivary gland make amylase to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylase. Specific amylase proteins are designated by different Greek letters. All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds.

<span class="mw-page-title-main">Cellulase</span> Class of enzymes

Cellulase is any of several enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharides:

<span class="mw-page-title-main">Maltase</span> Enzyme

Maltase is one type of alpha-glucosidase enzymes located in the brush border of the small intestine. This enzyme catalyzes the hydrolysis of disaccharide maltose into two simple sugars of glucose. Maltase is found in plants, bacteria, yeast, humans, and other vertebrates. It is thought to be synthesized by cells of the mucous membrane lining the intestinal wall.

<span class="mw-page-title-main">Dextrin</span> Chemical compound

Dextrins are a group of low-molecular-weight carbohydrates produced by the hydrolysis of starch and glycogen. Dextrins are mixtures of polymers of D-glucose units linked by α-(1→4) or α-(1→6) glycosidic bonds.

<span class="mw-page-title-main">Acarbose</span> Chemical compound

Acarbose (INN) is an anti-diabetic drug used to treat diabetes mellitus type 2 and, in some countries, prediabetes. It is a generic sold in Europe and China as Glucobay, in North America as Precose, and in Canada as Prandase. It is cheap and popular in China, but not in the U.S. One physician explains that use in the U.S. is limited because it is not potent enough to justify the side effects of diarrhea and flatulence. However, a large study concluded in 2013 that "acarbose is effective, safe and well tolerated in a large cohort of Asian patients with type 2 diabetes." A possible explanation for the differing opinions is an observation that acarbose is significantly more effective in patients eating a relatively high carbohydrate Eastern diet.

<span class="mw-page-title-main">Pullulanase</span> Enzyme

Pullulanase is a specific kind of glucanase, an amylolytic exoenzyme, that degrades pullulan. It is produced as an extracellular, cell surface-anchored lipoprotein by Gram-negative bacteria of the genus Klebsiella. Type I pullulanases specifically attack α-1,6 linkages, while type II pullulanases are also able to hydrolyse α-1,4 linkages. It is also produced by some other bacteria and archaea. Pullulanase is used as a processing aid in grain processing biotechnology.

<span class="mw-page-title-main">Glycogen debranching enzyme</span> Mammalian protein found in Homo sapiens

A debranching enzyme is a molecule that helps facilitate the breakdown of glycogen, which serves as a store of glucose in the body, through glucosyltransferase and glucosidase activity. Together with phosphorylases, debranching enzymes mobilize glucose reserves from glycogen deposits in the muscles and liver. This constitutes a major source of energy reserves in most organisms. Glycogen breakdown is highly regulated in the body, especially in the liver, by various hormones including insulin and glucagon, to maintain a homeostatic balance of blood-glucose levels. When glycogen breakdown is compromised by mutations in the glycogen debranching enzyme, metabolic diseases such as Glycogen storage disease type III can result.

Isomaltase is an enzyme that breaks the bonds linking saccharides, which cannot be broken by amylase or maltase. It digests polysaccharides at the alpha 1-6 linkages. Its substrate, alpha-limit dextrin, is a product of amylopectin digestion that retains its 1-6 linkage. The product of the enzymatic digestion of alpha-limit dextrin by isomaltase is maltose.

<span class="mw-page-title-main">Glycogen branching enzyme</span> Mammalian protein involved in glycogen production

1,4-alpha-glucan-branching enzyme, also known as brancher enzyme or glycogen-branching enzyme is an enzyme that in humans is encoded by the GBE1 gene.

β-Amylase Enzyme that hydrolyses alpha-1,4-D-glucosidic bonds in polysaccharides

β-Amylase is an enzyme with the systematic name 4-α-D-glucan maltohydrolase. It catalyses the following reaction:

<span class="mw-page-title-main">Maltotriose</span> Chemical compound

Maltotriose is a trisaccharide consisting of three glucose molecules linked with α-1,4 glycosidic bonds.

α-Amylase Enzyme that hydrolyses α bonds of large α-linked polysaccharides

α-Amylase is an enzyme that hydrolyses α bonds of large, α-linked polysaccharides, such as starch and glycogen, yielding shorter chains thereof, dextrins, and maltose:

<span class="mw-page-title-main">Cyclomaltodextrin glucanotransferase</span>

In enzymology, a cyclomaltodextrin glucanotransferase is an enzyme that catalyzes the chemical reaction of cyclizing part of a 1,4-alpha-D-glucan molecule through the formation of a 1,4-alpha-D-glucosidic bond. They are bacterial enzymes belonging to the same family of the α-amylase specifically known as glycosyl-hydrolase family 13. This peculiar enzyme is capable of catalyzing more than one reaction with the most important being the synthesis of non-reducing cyclic dextrins known as cyclodextrins starting from starch, amylose, and other polysaccharides.

<span class="mw-page-title-main">Alpha glucan</span>

α-Glucans (alpha-glucans) are polysaccharides of D-glucose monomers linked with glycosidic bonds of the alpha form. α-Glucans use cofactors in a cofactor site in order to activate a glucan phosphorylase enzyme. This enzyme causes a reaction that transfers a glucosyl portion between orthophosphate and α-I,4-glucan. The position of the cofactors to the active sites on the enzyme are critical to the overall reaction rate thus, any alteration to the cofactor site leads to the disruption of the glucan binding site.

<span class="mw-page-title-main">Glycoside hydrolase family 14</span>

In molecular biology, Glycoside hydrolase family 14 is a family of glycoside hydrolases.

<span class="mw-page-title-main">Glycoside hydrolase family 13</span>

In molecular biology, glycoside hydrolase family 13 is a family of glycoside hydrolases.

<span class="mw-page-title-main">Glucanase</span>

Glucanases are enzymes that break down large polysaccharides via hydrolysis. The product of the hydrolysis reaction is called a glucan, a linear polysaccharide made of up to 1200 glucose monomers, held together with glycosidic bonds. Glucans are abundant in the endosperm cell walls of cereals such as barley, rye, sorghum, rice, and wheat. Glucanases are also referred to as lichenases, hydrolases, glycosidases, glycosyl hydrolases, and/or laminarinases. Many types of glucanases share similar amino acid sequences but vastly different substrates. Of the known endo-glucanases, 1,3-1,4-β-glucanase is considered the most active.

Isomaltooligosaccharide (IMO) is a mixture of short-chain carbohydrates which has a digestion-resistant property. IMO is found naturally in some foods, as well as being manufactured commercially. The raw material used for manufacturing IMO is starch, which is enzymatically converted into a mixture of isomaltooligosaccharides.

Limit dextrinase is an enzyme with systematic name dextrin 6-alpha-glucanohydrolase. This enzyme catalyses the hydrolysis of (1->6)-alpha-D-glucosidic linkages in alpha- and beta-limits dextrins of amylopectin and glycogen, in amylopectin and pullulan.

<span class="mw-page-title-main">Glucan 1,4-alpha-maltohydrolase</span>

Glucan 1,4-alpha-maltohydrolase is an enzyme with systematic name 4-alpha-D-glucan alpha-maltohydrolase. This enzyme catalyses the following chemical reaction

References

  1. 1 2 Ohtaki, A; Mizuno, M; Tonozuka, T; Sakano, Y; Kamitori, S (2004). "Complex structures of Thermoactinomyces vulgaris R-47 alpha-amylase 2 with acarbose and cyclodextrins demonstrate the multiple substrate recognition mechanism". J Biol Chem. 279 (30): 31033–40. doi: 10.1074/jbc.M404311200 . PMID   15138257.
  2. 1 2 Imanaka T, Kuriki T (January 1989). "Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan". Journal of Bacteriology. 171 (1): 369–74. doi:10.1128/jb.171.1.369-374.1989. PMC   209598 . PMID   2914851.
  3. Takata, H; Kuriki, T; Okada, S; Takesada, Y; Iizuka, M; Minamiura, N; Imanaka, T (1992). "Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1----4)- and alpha-(1----6)-glucosidic linkages". J Biol Chem. 267 (26): 18447–52. PMID   1388153.
  4. Madan Babu, M. "TIM Barrel Analysis". MRC Laboratory of Molecular Biology. Anna University. Retrieved 5 March 2018.
  5. Hondoh H, Kuriki T, Matsuura Y (7 February 2003). "Three-dimensional Structure and Substrate Binding of Bacillus stearothermophilus Neopullulanase". Plant Molecular Biology. Journal of Molecular Biology, Volume 326, Issue 1, pp 177-188. 25: 141–157. doi:10.1007/BF00023233.
  6. 1 2 3 4 Svensson, Bert (May 1994). "Protein engineering in the α-amylase family: catalytic mechanism, substrate specificity, and stability". Plant Molecular Biology. 25 (2): 141–157. doi:10.1007/BF00023233.
  7. Rehm B.H.A (2009). Microbial production of biopolymers and polymers precursors. Caister Academic Press. p. 230.
  8. 1 2 Kuriki, T; Yanase, M; Takata, H; Takesada, Y; Imanaka, T; Okada, S (1993). "A new way of producing isomalto-oligosaccharide syrup by using the transglycosylation reaction of neopullulanase". Appl Environ Microbiol. 59 (4): 953–9. PMC   202222 . PMID   16348919.
  9. Minami, T; Miki, T; Fujiwara, T; Kawabata, Shigetada; Izumitani, A; Ooshima, T; Sobue, S; Hamada, Sherif (1989). "[Caries-inducing activity of isomaltooligosugar (IMOS) in in vitro and rat experiments]. Shōni shikagaku zasshi". The Japanese Journal of Pedodontics. 27: 1010–7.