Neopullulanase (, ''pullulanase II'') is an

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

of the alpha-amylase family with

systematic name A systematic name is a name given in a systematic way to one unique group, organism, object or chemical substance, out of a specific population or collection. Systematic names are usually part of a nomenclature. A semisystematic name or semitrivi ...

''pullulan 4-D-glucanohydrolase (panose-forming)''. This enzyme principally catalyses the following

chemical reaction A chemical reaction is a process that leads to the chemistry, chemical transformation of one set of chemical substances to another. When chemical reactions occur, the atoms are rearranged and the reaction is accompanied by an Gibbs free energy, ...

by cleaving pullulan's alpha-1,4-glucosidic bonds: : Hydrolysis of

pullulan Pullulan is a polysaccharide consisting of maltotriose units, also known as α-1,4- ;α-1,6-glucan'. Three glucose units in maltotriose are connected by an α-1,4 glycosidic bond, whereas consecutive maltotriose units are connected to each other ...

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 } Maltose ( or ), also known as maltobiose or malt sugar, is a disaccharide formed from two units of glucose joined with an α(1→4) bond. In the isomer isomaltose, the two glucose molecules are joined with an α(1→6) bond. Maltose is the tw ...

and

glucose Glucose is a sugar with the Chemical formula#Molecular formula, molecular formula , which is often abbreviated as Glc. It is overall the most abundant monosaccharide, a subcategory of carbohydrates. It is mainly made by plants and most algae d ...

Structure

Neopullulanase is a

dimer Dimer may refer to: * Dimer (chemistry), a chemical structure formed from two similar sub-units ** Protein dimer, a protein quaternary structure ** d-dimer ** TH-dimer * Dimer model, an item in statistical mechanics, based on ''domino tiling'' * ...

of identical monomer subunits, each with four domains (N,A,B,C) that are highly conserved with other starch hydrolases, namely alpha-amylase,

pullulanase Pullulanase (, ''limit dextrinase'', ''amylopectin 6-glucanohydrolase'', ''bacterial debranching enzyme'', ''debranching enzyme'', ''α-dextrin endo-1,6-α-glucosidase'', ''R-enzyme'', ''pullulan α-1,6-glucanohydrolase'') is a specific kind of g ...

cyclomaltodextrin glucanotransferase In enzymology, a cyclomaltodextrin glucanotransferase (also cyclodextrin glycosyl transferase or CGTase for short) () is an enzyme that catalyzes the chemical reaction of cyclizing part of a 1,4-alpha-D-glucan molecule through the formation of a ...

, and 1,4-alpha-D-glucan branching enzyme (also known as

glycogen branching enzyme 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. Glycogen branching enzyme is an enzyme that adds branches to the growing glycogen molecu ...

). Like these enzymes, each monomer contains an

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...

at the carboxyl-terminus within a

TIM barrel The TIM barrel (triose-phosphate isomerase), also known as an alpha/beta barrel, is a conserved protein fold consisting of eight alpha helices (α-helices) and eight parallel beta strands (β-strands) that alternate along the peptide backbone. ...

(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. 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 Sequence homology is the homology (biology), biological homology between DNA sequence, DNA, RNA sequence, RNA, or Protein primary structure, protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments ...

. 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.

Mechanism

The hydrolysis of pullulan to panose is catalyzed by three

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

residues within neopullulanase's active site that cleave a glycosidic bond: one

glutamate Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a Essential amino acid, non-essential nutrient for humans, meaning that ...

and two

aspartate Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protein ...

s. A glycosidic oxygen is first protonated by the

carboxyl group In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is often written as or , sometimes as with R referring to an organyl group (e.g. ...

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 In organic chemistry, a carboxylate is the conjugate base of a carboxylic acid, (or ). It is an anion, an ion with negative charge. Carboxylate salts are salts that have the general formula , where M is a metal and ''n'' is 1, 2,... ...

group of a second aspartate residue (TAA Asp-297) deprotonates an adjacent water molecule to form a

hydroxide ion Hydroxide is a polyatomic ion, diatomic anion with chemical formula OH−. It consists of an oxygen and hydrogen atom held together by a single covalent bond, and carries a negative electric charge. It is an important but usually Self-ionization ...

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 In chemistry, hydroxylation refers to the installation of a hydroxyl group () into an organic compound. Hydroxylations generate alcohols and phenols, which are very common functional groups. Hydroxylation confers some degree of water-solubility ...

The three residues responsible for neopullulanase catalysis are invariably present in enzymes of the alpha-amylase family. 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. 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 Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an Amine, α-amino group (which is in the protonated –NH3+ form under Physiological condition, biological conditions), a carboxylic ...

residues (TAA His-122 and TAA His-296) that interact with the

glycan The terms glycans and polysaccharides are defined by IUPAC as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". However, in practice the term glycan may also be used to refer to the carbohydrate ...

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 Polysaccharides (), or polycarbohydrates, are the most abundant carbohydrates found in food. They are long-chain polymeric carbohydrates composed of monosaccharide units bound together by glycosidic linkages. This carbohydrate can react with wat ...

polymer A polymer () is a chemical substance, substance or material that consists of very large molecules, or macromolecules, that are constituted by many repeat unit, repeating subunits derived from one or more species of monomers. Due to their br ...

consisting of repeating

maltotriose Maltotriose is a trisaccharide (three-part sugar) consisting of three glucose molecules linked with α-1,4 glycosidic bonds. It is most commonly produced by the digestive enzyme alpha-amylase (a common enzyme in human saliva) on amylose in star ...

units. It provides a protective effect against cellular

desiccation Desiccation is the state of extreme dryness, or the process of extreme drying. A desiccant is a hygroscopic (attracts and holds water) substance that induces or sustains such a state in its local vicinity in a moderately sealed container. The ...

in low-moisture environments. 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 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 starc ...

syrup using ''

Bacillus stearothermophilus ''Geobacillus stearothermophilus'' (previously ''Bacillus stearothermophilus'') is a rod-shaped, Gram-positive bacterium and a member of the phylum Bacillota. The bacterium is a thermophile and is widely distributed in soil, hot springs, ocean se ...

'' neopullulase has been proposed, taking advantage of neopullulase's ability to catalyze hydrolysis of branched oligosaccharides' alpha-1-6-glucosidic linkages. 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. 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 ''Bacillus subtilis'' (), known also as the hay bacillus or grass bacillus, is a gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacill ...

'' may be added to the solution.

References

External links