ATP Hydrolysis by the SNF2 Domain of Dnmt5 Is Coupled to Both Specific Recognition and Modification of Hemimethylated DNA.

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step essential for DNA methylation. Coupling occurs independent of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating Dnmt5's ATP-binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a "flipped-out" conformation of the target cytosine bypass the SNF2 domain's requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to the DNMT domain conformational changes induced by preferred substrates. These findings establish a new role for a SNF2 ATPase: controlling an adjoined enzymatic domain's substrate recognition and catalysis. We speculate that this coupling contributes to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.

Metabolite Repair Enzymes Control Metabolic Damage in Glycolysis.

Hundreds of metabolic enzymes work together smoothly in a cell. These enzymes are highly specific. Nevertheless, under physiological conditions, many perform side-reactions at low rates, producing potentially toxic side-products. An increasing number of metabolite repair enzymes are being discovered that serve to eliminate these noncanonical metabolites. Some of these enzymes are extraordinarily conserved, and their deficiency can lead to diseases in humans or embryonic lethality in mice, indicating their central role in cellular metabolism. We discuss how metabolite repair enzymes eliminate glycolytic side-products and prevent negative interference within and beyond this core metabolic pathway. Extrapolating from the number of metabolite repair enzymes involved in glycolysis, hundreds more likely remain to be discovered that protect a wide range of metabolic pathways.

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases.

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.

Support Enzyme Loading Influences the Effect of Aldehyde Dextran Modification on the Specificity of Immobilized Ficin for Large Proteins.

It has been reported that the modification of immobilized glyoxyl-ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl-ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking).

DNA methyltransferase DNMT3A forms interaction networks with the CpG site and flanking sequence elements for efficient methylation.

Specific DNA methylation at CpG and non-CpG sites is essential for chromatin regulation. The DNA methyltransferase DNMT3A interacts with target sites surrounded by variable DNA sequences with its TRD and RD loops, but the functional necessity of these interactions is unclear. We investigated CpG and non-CpG methylation in a randomized sequence context using WT DNMT3A and several DNMT3A variants containing mutations at DNA-interacting residues. Our data revealed that the flanking sequence of target sites between the -2 and up to the +8 position modulates methylation rates >100-fold. Non-CpG methylation flanking preferences were even stronger and favor C(+1). R836 and N838 in concert mediate recognition of the CpG guanine. R836 changes its conformation in a flanking sequence-dependent manner and either contacts the CpG guanine or the +1/+2 flank, thereby coupling the interaction with both sequence elements. R836 suppresses activity at CNT sites but supports methylation of CAC substrates, the preferred target for non-CpG methylation of DNMT3A in cells. N838 helps to balance this effect and prevent the preference for C(+1) from becoming too strong. Surprisingly, we found L883 reduces DNMT3A activity despite being highly conserved in evolution. However, mutations at L883 disrupt the DNMT3A-specific DNA interactions of the RD loop, leading to altered flanking sequence preferences. Similar effects occur after the R882H mutation in cancer cells. Our data reveal that DNMT3A forms flexible and interdependent interaction networks with the CpG guanine and flanking residues that ensure recognition of the CpG and efficient methylation of the cytosine in contexts of variable flanking sequences.

[Mechanisms of the Specificity of the CRISPR/Cas9 System in Genome Editing].

The CRISPR/Cas9 system, which was discovered recently, utilizes nucleases targeted by sequence complementarity and is originally intended to protect bacteria from foreign genetic elements. The system provided a convenient tool for manipulating the genomes of living cells. The CRISPR/Cas9 genomic editing technology moved beyond the laboratory and already found application in biotechnology and agriculture. However, off-target activity of the CRISPR/Cas9 system can cause oncogenic mutations and thus limits its use for genome editing in human cells for medical purposes. Many studies are therefore aimed at developing variants of the CRISPR/Cas9 system with improved accuracy. The review considers the mechanisms of precise and erroneous actions of Cas9 RNA-guided nuclease, natural and artificial variants of RNA-targeted nucleases, possibilities to modulate their specificity through guide RNA modifications, and other approaches to increasing the accuracy of the CRISPR/Cas9 system in genome editing.

Homing in: Mechanisms of Substrate Targeting by Protein Kinases.

Protein phosphorylation is the most common reversible post-translational modification in eukaryotes. Humans have over 500 protein kinases, of which more than a dozen are established targets for anticancer drugs. All kinases share a structurally similar catalytic domain, yet each one is uniquely positioned within signaling networks controlling essentially all aspects of cell behavior. Kinases are distinguished from one another based on their modes of regulation and their substrate repertoires. Coupling specific inputs to the proper signaling outputs requires that kinases phosphorylate a limited number of sites to the exclusion of hundreds of thousands of off-target phosphorylation sites. Here, we review recent progress in understanding mechanisms of kinase substrate specificity and how they function to shape cellular signaling networks.

Active site architecture reveals coordination sphere flexibility and specificity determinants in a group of closely related molybdoenzymes.

MtsZ is a molybdenum-containing methionine sulfoxide reductase that supports virulence in the human respiratory pathogen Haemophilus influenzae (Hi). HiMtsZ belongs to a group of structurally and spectroscopically uncharacterized S-/N-oxide reductases, all of which are found in bacterial pathogens. Here, we have solved the crystal structure of HiMtsZ, which reveals that the HiMtsZ substrate-binding site encompasses a previously unrecognized part that accommodates the methionine sulfoxide side chain via interaction with His182 and Arg166. Charge and amino acid composition of this side chain-binding region vary and, as indicated by electrochemical, kinetic, and docking studies, could explain the diverse substrate specificity seen in closely related enzymes of this type. The HiMtsZ Mo active site has an underlying structural flexibility, where dissociation of the central Ser187 ligand affected catalysis at low pH. Unexpectedly, the two main HiMtsZ electron paramagnetic resonance (EPR) species resembled not only a related dimethyl sulfoxide reductase but also a structurally unrelated nitrate reductase that possesses an Asp-Mo ligand. This suggests that contrary to current views, the geometry of the Mo center and its primary ligands, rather than the specific amino acid environment, is the main determinant of the EPR properties of mononuclear Mo enzymes. The flexibility in the electronic structure of the Mo centers is also apparent in two of three HiMtsZ EPR-active Mo(V) species being catalytically incompetent off-pathway forms that could not be fully oxidized.

GH18 endo-ß-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-ß-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis.

Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. An increase of the Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases.

Immobilization of Lipase B from Candida antarctica in Octyl-Vinyl Sulfone Agarose: Effect of the Enzyme-Support Interactions on Enzyme Activity, Specificity, Structure and Inactivation Pathway.

Lipase B from Candida antarctica was immobilized on heterofunctional support octyl agarose activated with vinyl sulfone to prevent enzyme release under drastic conditions. Covalent attachment was established, but the blocking step using hexylamine, ethylenediamine or the amino acids glycine (Gly) and aspartic acid (Asp) altered the results. The activities were lower than those observed using the octyl biocatalyst, except when using ethylenediamine as blocking reagent and p-nitrophenol butyrate (pNPB) as substrate. The enzyme stability increased using these new biocatalysts at pH 7 and 9 using all blocking agents (much more significantly at pH 9), while it decreased at pH 5 except when using Gly as blocking agent. The stress inactivation of the biocatalysts decreased the enzyme activity versus three different substrates (pNPB, S-methyl mandelate and triacetin) in a relatively similar fashion. The tryptophane (Trp) fluorescence spectra were different for the biocatalysts, suggesting different enzyme conformations. However, the fluorescence spectra changes during the inactivation were not too different except for the biocatalyst blocked with Asp, suggesting that, except for this biocatalyst, the inactivation pathways may not be so different.

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts.

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme®435 (L435) (lipase B from Candida antarctica), Lipozyme®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.

A predicted transmembrane region in plant diacylglycerol acyltransferase 2 regulates specificity toward very-long-chain acyl-CoAs.

Triacylglycerols are the main constituent of seed oil. The specific fatty acid composition of this oil is strongly impacted by the substrate specificities of acyltransferases involved in lipid synthesis, such as the integral membrane enzyme diacylglycerol acyltransferase (DGAT). Two forms of DGAT, DGAT1 and DGAT2, are thought to contribute to the formation of seed oil, and previous characterizations of various DGAT2 enzymes indicate that these often are associated with the incorporation of unusual fatty acids. However, the basis of DGAT2's acyl-donor specificity is not known because of the inherent challenges of predicting structural features of integral membrane enzymes. The recent characterization of DGAT2 enzymes from Brassica napus reveals that DGAT2 enzymes with similar amino acid sequences exhibit starkly contrasting acyl-donor specificities. Here we have designed and biochemically tested a range of chimeric enzymes, substituting parts of these B. napus DGAT2 enzymes with each other, allowing us to pinpoint a region that dramatically affects the specificity toward 22:1-CoA. It may thus be possible to redesign the acyl-donor specificity of DGAT2 enzymes, potentially altering the fatty acid composition of seed oil. Further, the characterization of a DGAT2 chimera between Arabidopsis and B. napus demonstrates that the specificity regulated by this region is transferrable across species. The identified region contains two predicted transmembrane helices that appear to reoccur in a wide range of plant DGAT2 orthologues, suggesting that it is a general feature of plant DGAT2 enzymes.

Machine learning-based prediction of enzyme substrate scope: Application to bacterial nitrilases.

Predicting the range of substrates accepted by an enzyme from its amino acid sequence is challenging. Although sequence- and structure-based annotation approaches are often accurate for predicting broad categories of substrate specificity, they generally cannot predict which specific molecules will be accepted as substrates for a given enzyme, particularly within a class of closely related molecules. Combining targeted experimental activity data with structural modeling, ligand docking, and physicochemical properties of proteins and ligands with various machine learning models provides complementary information that can lead to accurate predictions of substrate scope for related enzymes. Here we describe such an approach that can predict the substrate scope of bacterial nitrilases, which catalyze the hydrolysis of nitrile compounds to the corresponding carboxylic acids and ammonia. Each of the four machine learning models (logistic regression, random forest, gradient-boosted decision trees, and support vector machines) performed similarly (average ROC = 0.9, average accuracy = ~82%) for predicting substrate scope for this dataset, although random forest offers some advantages. This approach is intended to be highly modular with respect to physicochemical property calculations and software used for structural modeling and docking.

Underground metabolism facilitates the evolution of novel pathways for vitamin B6 biosynthesis.

The term vitamin B6 is a designation for the vitamers pyridoxal, pyridoxamine, pyridoxine and the respective phosphate esters pyridoxal-5'-phosphate (PLP), pyridoxamine-5'-phosphate and pyridoxine-5'-phosphate. Animals and humans are unable to synthesise vitamin B6. These organisms have to take up vitamin B6 with their diet. Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry. As yet, two naturally occurring routes for de novo synthesis of PLP are known. Both routes have been genetically engineered to obtain bacteria overproducing vitamin B6. Still, major genetic engineering efforts using the existing pathways are required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Recent suppressor screens using mutants of the Gram-negative and Gram-positive model bacteria Escherichia coli and Bacillus subtilis, respectively, carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis. These pathways consist of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. Thus, E. coli and B. subtilis contain multiple promiscuous enzymes causing a so-called underground metabolism allowing the bacteria to bypass disrupted vitamin B6 biosynthetic pathways. The suppressor screens also show the genomic plasticity of the bacteria to suppress a genetic lesion. We discuss the potential of the serendipitous pathways to serve as a starting point for the development of bacteria overproducing vitamin B6. KEY POINTS: • Known vitamin B6 routes have been genetically engineered. • Underground metabolism facilitates the emergence of novel vitamin B6 biosynthetic pathways. • These pathways may be suitable to engineer bacteria overproducing vitamin B6.

Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity.

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer's, Parkinson's, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

Loops around the Heme Pocket Have a Critical Role in the Function and Stability of BsDyP from Bacillus subtilis.

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme's overall stability by 2 kcal mol-1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.

Enzymatic basis for C-lignin monomer biosynthesis in the seed coat of Cleome hassleriana.

C-lignin is a linear polymer of caffeyl alcohol, found in the seed coats of several exotic plant species, with promising properties for generation of carbon fibers and high value chemicals. In the ornamental plant Cleome hassleriana, guaiacyl (G) lignin is deposited in the seed coat for the first 6-12 days after pollination, after which G-lignin deposition ceases and C-lignin accumulates, providing an excellent model system to study C-lignin biosynthesis. We performed RNA sequencing of seed coats harvested at 2-day intervals throughout development. Bioinformatic analysis identified a complete set of lignin biosynthesis genes for Cleome. Transcript analysis coupled with kinetic analysis of recombinant enzymes in Escherichia coli revealed that the switch to C-lignin formation was accompanied by down-regulation of transcripts encoding functional caffeoyl CoA- and caffeic acid 3-O-methyltransferases (CCoAOMT and COMT) and a form of cinnamyl alcohol dehydrogenase (ChCAD4) with preference for coniferaldehyde as substrate, and up-regulation of a form of CAD (ChCAD5) with preference for caffealdehyde. Based on these analyses, blockage of lignin monomer methylation by down-regulation of both O-methyltransferases (OMTs) and methionine synthase (for provision of C1 units) appears to be the major factor in diversion of flux to C-lignin in the Cleome seed coat, although the change in CAD specificity also contributes based on the reduction of C-lignin levels in transgenic Cleome with down-regulation of ChCAD5. Structure modeling and mutational analysis identified amino acid residues important for the preference of ChCAD5 for caffealdehyde.

Analysis of the Substrate Specificity of the SMYD2 Protein Lysine Methyltransferase and Discovery of Novel Non-Histone Substrates.

The SMYD2 protein lysine methyltransferase methylates various histone and non-histone proteins and is overexpressed in several cancers. Using peptide arrays, we investigated the substrate specificity of the enzyme, revealing a recognition of leucine (or weaker phenylalanine) at the -1 peptide site and disfavor of acidic residues at the +1 to +3 sites. Using this motif, novel SMYD2 peptide substrates were identified, leading to the discovery of 32 novel peptide substrates with a validated target site. Among them, 19 were previously reported to be methylated at the target lysine in human cells, strongly suggesting that SMYD2 is the protein lysine methyltransferase responsible for this activity. Methylation of some of the novel peptide substrates was tested at the protein level, leading to the identification of 14 novel protein substrates of SMYD2, six of which were more strongly methylated than p53, the best SMYD2 substrate described so far. The novel SMYD2 substrate proteins are involved in diverse biological processes such as chromatin regulation, transcription, and intracellular signaling. The results of our study provide a fundament for future investigations into the role of this important enzyme in normal development and cancer.

Cloning and characterization of a novel amylopullulanase from Bacillus megaterium Y103 with transglycosylation activity.

OBJECTIVE: To obtain a novel pullulanase with synthetic ability from a microorganism and characterize its substrates specificity. RESULTS: A novel pullulanase, PulY103A, from Bacillus megaterium Y103 was purified, characterized and expressed in Escherichia coli. PulY103A contained the signature sequences of type I pullulanases and showed 94.7% identity with a type I pullulanase (BmPul) from B. megaterium WW1210, showing similar molecular weight (110.8 kDa) and optimal pH (6.5). However, PulY103A had an optimal temperature of of 45 °C and exhibited relatively higher activity toward amylose (48.3%) compared with pullulan (100%), soluble starch (67.5%), and amylopectin (23.1%). The thin-layer chromatography results showed that the major pullulan hydrolysis products were maltotriose and maltohexaose, which differed from those reported in other pullulanases. On the basis of enzyme specificity, PulY103A was an amylopullulanase, which presented transglycosylation activity by forming α-1,4-glucosidic linkages. CONCLUSIONS: A novel amylopullulanase with transglycosylation activity was characterized. The features of this enzyme suggested its potential to produce maltohexaose.