R-hydroxynitrile lyase from the cyanogenic millipede, Chamberlinius hualienensis-A new entry to the carrier protein family Lipocalines.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrin into cyanide and the corresponding aldehyde or ketone. Moreover, they catalyze the synthesis of cyanohydrin in the reverse reaction, utilized in industry for preparation of enantiomeric pure pharmaceutical ingredients and fine chemicals. We discovered a new HNL from the cyanogenic millipede, Chamberlinius hualienensis. The enzyme displays several features including a new primary structure, high stability, and the highest specific activity in (R)-mandelonitrile ((R)-MAN) synthesis (7420 U·mg-1 ) among the reported HNLs. In this study, we elucidated the crystal structure and reaction mechanism of natural ChuaHNL in ligand-free form and its complexes with acetate, cyanide ion, and inhibitors (thiocyanate or iodoacetate) at 1.6, 1.5, 2.1, 1.55, and 1.55 Å resolutions, respectively. The structure of ChuaHNL revealed that it belongs to the lipocalin superfamily, despite low amino acid sequence identity. The docking model of (R)-MAN with ChuaHNL suggested that the hydroxyl group forms hydrogen bonds with R38 and K117, and the nitrile group forms hydrogen bonds with R38 and Y103. The mutational analysis showed the importance of these residues in the enzymatic reaction. From these results, we propose that K117 acts as a base to abstract a proton from the hydroxyl group of cyanohydrins and R38 acts as an acid to donate a proton to the cyanide ion during the cleavage reaction of cyanohydrins. The reverse mechanism would occur during the cyanohydrin synthesis. (Photo: Dr. Yuko Ishida) DATABASES: Structural data are available in PDB database under the accession numbers 6JHC, 6KFA, 6KFB, 6KFC, and 6KFD.

Improvement of ST0452 N-Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering.

We showed previously that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibited over 4 times higher N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. The same interaction was observed for Escherichia coli GlmU in the absence of metal ions. These observations indicated that the three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein also showed that positions 146 (Glu) and 80 (Thr) formed interactions with GlcNAc, and an engineering strategy was applied to these residues to increase activity. All proteins substituted at position 146 had drastically decreased activities, whereas several proteins substituted at position 80 showed higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein. The substituted amino acids at positions 80 and 97 might result in optimized interactions with the substrate; therefore, we predicted that the combination of these two substitutions might cooperatively increase GlcNAc-1-P UTase activity. Of the four double mutant ST0452 proteins generated, T80S/Y97N showed 6.5-times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues functioned cooperatively to increase GlcNAc-1-P UTase activity.IMPORTANCE We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes.

Identification of a Direct Biosynthetic Pathway for UDP-N-Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii.

Most organisms, from Bacteria to Eukarya, synthesize UDP-N-acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP-N-acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea, the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii, predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii, and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways.IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially.

Rational identification of aggregation hotspots based on secondary structure and amino acid hydrophobicity.

Insolubility of proteins expressed in the Escherichia coli expression system hinders the progress of both basic and applied research. Insoluble proteins contain residues that decrease their solubility (aggregation hotspots). Mutating these hotspots to optimal amino acids is expected to improve protein solubility. To date, however, the identification of these hotspots has proven difficult. In this study, using a combination of approaches involving directed evolution and primary sequence analysis, we found two rules to help inductively identify hotspots: the α-helix rule, which focuses on the hydrophobicity of amino acids in the α-helix structure, and the hydropathy contradiction rule, which focuses on the difference in hydrophobicity relative to the corresponding amino acid in the consensus protein. By properly applying these two rules, we succeeded in improving the probability that expressed proteins would be soluble. Our methods should facilitate research on various insoluble proteins that were previously difficult to study due to their low solubility.

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent kcat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. IMPORTANCE: It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent kcat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity.

A sacrificial millipede altruistically protects its swarm using a drone blood enzyme, mandelonitrile oxidase.

Soldiers of some eusocial insects exhibit an altruistic self-destructive defense behavior in emergency situations when attacked by large enemies. The swarm-forming invasive millipede, Chamberlinius hualienensis, which is not classified as eusocial animal, exudes irritant chemicals such as benzoyl cyanide as a defensive secretion. Although it has been thought that this defensive chemical was converted from mandelonitrile, identification of the biocatalyst has remained unidentified for 40 years. Here, we identify the novel blood enzyme, mandelonitrile oxidase (ChuaMOX), which stoichiometrically catalyzes oxygen consumption and synthesis of benzoyl cyanide and hydrogen peroxide from mandelonitrile. Interestingly the enzymatic activity is suppressed at a blood pH of 7, and the enzyme is segregated by membranes of defensive sacs from mandelonitrile which has a pH of 4.6, the optimum pH for ChuaMOX activity. In addition, strong body muscle contractions are necessary for de novo synthesis of benzoyl cyanide. We propose that, to protect its swarm, the sacrificial millipede also applies a self-destructive defense strategy-the endogenous rupturing of the defensive sacs to mix ChuaMOX and mandelonitrile at an optimum pH. Further study of defensive systems in primitive arthropods will pave the way to elucidate the evolution of altruistic defenses in the animal kingdom.

Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis.

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

Structural and functional analysis of hydroxynitrile lyase from Baliospermum montanum with crystal structure, molecular dynamics and enzyme kinetics.

Hydroxynitrile lyases (HNLs) catalyze degradation of cyanohydrins to hydrogen cyanide and the corresponding ketone or aldehyde. HNLs can also catalyze the reverse reaction, i.e., synthesis of cyanohydrins. Although several crystal structures of S-selective hydroxynitrile lyases (S-HNLs) have been reported, it remains unknown whether and how dynamics at the active site of S-HNLs influence their broad substrate specificity and affinity. In this study, we analyzed the structure, dynamics and function of S-HNL from Baliospermum montanum (BmHNL), which has an α/ß hydrolase fold. Two crystal structures of BmHNL, apo1 and apo2, were determined at 2.55 and 1.9Å, respectively. Structural comparison between BmHNL (apo2) and S-HNL from Hevea brasiliensis with (S)-mandelonitrile bound to the active site revealed that hydrophobic residues at the entrance region of BmHNL formed hydrophobic interactions with the benzene ring of the substrate. The flexible structures of these hydrophobic residues were confirmed by a 15ns molecular dynamics simulation. This flexibility regulated the size of the active site cavity, enabling binding of various substrates to BmHNL. The high affinity of BmHNL toward substrates containing a benzene ring was also confirmed by comparing the kinetics of BmHNL and S-HNL from Manihot esculenta. Taken together, the results indicated that the flexibility and placement of the residues are important for the broad substrate specificity of S-HNLs.

Functional expression of a plant hydroxynitrile lyase in Escherichia coli by directed evolution: creation and characterization of highly in vivo soluble mutants.

Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering. Structure-guided saturation mutagenesis caused high solubility of single Lys-Pro mutations at positions 176, 199 and 224 of this low soluble wild-type enzyme. The triple Lys-Pro mutant generated at these surface conserved residues showed up to 8-fold increase in specific activity in the cell-free extract. Random mutagenesis also created a mutant of His103Met with 18.5-fold increase. The main expression form was reversed from insoluble to the soluble fraction following both types of above-mentioned mutations in E.coli at 37°C. The findings challenge the rationale of producing recombinant proteins in this host at 37°C. Formerly wild type low soluble protein was then present as soluble protein by these mutations, which also elevated the total soluble protein fraction in E.coli. Saturation mutagenesis of His103 provided other highly soluble mutants with hydrophobic substitutions. These mutations caused only minor secondary structural changes as determined by circular dichroism and Fourier-transform infrared spectroscopy and affected catalytic efficiency slightly for the purified mutants (0.82-1.6-fold for benzaldehyde and 0.9-1.9-fold for mandelonitrile). The stability of the mutants was differed from that of the wild type at high temperatures and at pH >8. Exchanging the buried basic-polar residue His103 with hydrophobic amino acids is in line with the overall structure of the enzyme, i.e. having hydrophilic residues in solvent-exposed areas and hydrophobic residues in the core.

S-selective hydroxynitrile lyase from a plant Baliospermum montanum: molecular characterization of recombinant enzyme.

A novel S-hydroxynitrile lyase (HNL) was purified from leaves of a plant, Baliospermum montanum, by ammonium sulfate fractionation and column chromatographies. Full-length cDNA and genomic DNA were cloned and sequenced. The latter contained two introns and one ORF encoding a 263-residue protein (subunit: 29.5 kDa). The hnl gene was expressed in Escherichia coli and the enzyme was characterized including detailed kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. The enzyme exhibited the highest specific activity (178 U/mg), k(cat) (98/s) and k(cat)/K(m) ratio for piperonal. k(cat)/K(m) ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO3, PMSF, phenol and methyl ethyl ketone, showed an optimum at pH 5, while having activity at range of 4-6.5. It exhibited stability at wide pH range 2.4-11, the highest activity at 20 °C, being active at 0-65 °C. The enzyme showed variations in residues involved in substrate pocket and substrate entrance channel compared to other S-selective HNLs, based on a model was built. C-terminal short truncations provided more enzyme production. Gel filtration revealed a 60-65 kDa molecular mass for this non-FAD enzyme and its C-terminally truncated forms using three buffer compositions, indicating dimeric structures.

Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems.

Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too.