Design and Construction of an Effective Expression System with Aldehyde Tag for Site-Specific Enzyme Immobilization

In recent years, the development and application of site-specific immobilization technology for proteins have undergone significant advances, which avoids the unwanted and random covalent linkage between the support and active site of protein in the covalent immobilization. Formylglycine generating enzyme (FGE) can transform the cysteine from a conversed 6-amino-acid sequence CXPXR into formylglycine with an aldehyde group (also termed as “aldehyde tag”). Based on the frame of pET-28a, the His-tags were replaced with aldehyde tags. Afterward, a set of plasmids were constructed for site-specific covalent immobilization, their His-tags were knock out (DH), or were replaced at different positions: N-terminal (NQ), C-terminal (CQ), or both (DQ) respectively. Three different enzymes, thermophilic acyl aminopeptidase (EC 3.4.19.1) from Sulfolobus tokodaii (ST0779), thermophilic dehalogenase (EC 3.8.1.2) from Sulfolobus tokodaii (ST2570), and Lipase A (EC 3.1.1.3) from Bacillus subtilis (BsLA) were chosen as model enzymes to connect with these plasmid systems. The results showed that different aldehyde-tagged enzymes can be successfully covalently attached to different carriers modified with an amino group, proving the universality of the method. The new immobilized enzyme also presented better thermostability and reutilization than those of the free enzyme.

Crystal Structures of the Putative Isocitrate Dehydrogenase from Sulfolobus tokodaii Strain 7 in the Apo and NADP+-Bound Forms

Isocitrate dehydrogenase is a catabolic enzyme that acts during the third step of the tricarboxylic acid cycle. The hypothetical protein ST2166 from the archaeon Sulfolobus tokodaii was isolated and crystallized. It shares high primary structure homology with prokaryotic NADP+-dependent IDHs, suggesting that these enzymes share a common enzymatic mechanism. The crystal structure of ST2166 was determined at 2.0 Å resolution in the apo form, and then the structure of the crystal soaked with NADP+ was also determined at 2.4 Å resolution, which contained NADP+ bound at the putative active site. Comparisons between the structures of apo and NADP+-bound forms and NADP-IDHs from other prokaryotes suggest that prokaryotic NADP-IDHs recognize their cofactors using conserved Lys335, Tyr336, and Arg386 in ST2166 at the opening cleft before the domain closure.

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

ABSTRACT 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.