Characterization and Crystal Structures of a Cubebol-Producing Sesquiterpene Synthase from Antrodia cinnamomea.

Antrodia cinnamomea is an endemic species found in Taiwan, known for its medicinal properties in treating various discomforts, including inflammation, diarrhea, abdominal pain, and other diseases. A. cinnamomea contains terpenoids that exhibit numerous bioactivities, making them potential food additives. This discovery piqued our interest in uncovering their biosynthetic pathway. Herein, we conducted functional and structural characterization of a sesquiterpene synthase Cop4 from A. cinnamomea (AcCop4). Through gas chromatography-mass spectrometry analysis, we observed that AcCop4 catalyzes the cyclization of farnesyl pyrophosphate (FPP), primarily producing cubebol. Cubebol is widely used as a long-lasting cooling and refreshing agent in the food industry. The structure of AcCop4, complexed with pyrophosphate and magnesium ions, revealed the closure of the active site facilitated by R311. Interestingly, binding of pyrophosphate and magnesium ions did not cause any significant conformational change in the G1/2 helix of AcCop4, indicating that the apo form is not fully open. This high-resolution structure serves as a solid basis for understanding the biosynthetic mechanism of AcCop4 and supports further production and modification of cubebol for its applications in the food industry.

Crystal structures of Aspergillus oryzae Rib2 deaminase: the functional mechanism involved in riboflavin biosynthesis.

Riboflavin serves as the direct precursor of the FAD/FMN coenzymes and is biosynthesized in most prokaryotes, fungi and plants. Fungal Rib2 possesses a deaminase domain for deamination of pyrimidine in the third step of riboflavin biosynthesis. Here, four high-resolution crystal structures of a Rib2 deaminase from Aspergillus oryzae (AoRib2) are reported which display three distinct occluded, open and complex forms that are involved in substrate binding and catalysis. In addition to the deaminase domain, AoRib2 contains a unique C-terminal segment which is rich in charged residues. Deletion of this unique segment has no effect on either enzyme activity or protein stability. Nevertheless, the C-terminal αF helix preceding the segment plays a role in maintaining protein stability and activity. Unexpectedly, AoRib2 is the first mononucleotide deaminase found to exist as a monomer, perhaps due to the assistance of its unique longer loops (Lß1-ß2, LαB-ß3 and LαC-ß4). These results form the basis for a molecular understanding of riboflavin biosynthesis in fungi and might assist in the development of antibiotics.

The crystal structure of protein-transporting chaperone BCP1 from Saccharomyces cerevisiae.

BCP1 is a protein enriched in the nucleus that is required for Mss4 nuclear export and identified as the chaperone of ribosomal protein Rpl23 in Saccharomyces cerevisiae. According to sequence homology, BCP1 is related to the mammalian BRCA2-interacting protein BCCIP and belongs to the BCIP protein family (PF13862) in the Pfam database. However, the BCIP family has no discernible similarity to proteins with known structure. Here, we report the crystal structure of BCP1, presenting an α/ß fold in which the central antiparallel ß-sheet is flanked by helices. Protein structural classification revealed that BCP1 has similarity to the GNAT superfamily but no conserved substrate-binding residues. Further modeling and protein-protein docking work provide a plausible model to explain the interaction between BCP1 and Rpl23. Our structural analysis presents the first structure of BCIP family and provides a foundation for understanding the molecular basis of BCP1 as a chaperone of Rpl23 for ribosome biosynthesis.

Crystal Structure of α-Galactosidase from Thermus thermophilus: Insight into Hexamer Assembly and Substrate Specificity.

α-Galactosidase catalyzes the hydrolysis of a terminal α-galactose residue in galacto-oligosaccharides and has potential in various industrial applications and food processing. We determined the crystal structures of α-galactosidase from the thermophilic microorganism Thermus thermophilus (TtGalA) and its complexes with pNPGal and stachyose. The monomer folds into an N-terminal domain, a catalytic (ß/α)8 barrel domain, and a C-terminal domain. The domain organization is similar to that of the other family of 36 α-galactosidases, but TtGalA presents a cagelike hexamer. Structural analysis shows that oligomerization may be a key factor for the thermal adaption of TtGalA. The structure of TtGalA complexed with stachyose reveals only the existence of one -1 subsite and one +1 subsite in the active site. Structural comparison of the stachyose-bound complexes of TtGalA and GsAgaA, a tetrameric enzyme with four subsites, suggests evolutionary divergence of substrate specificity within the GH36 family of α-galactosidases. To the best of our knowledge, the crystal structure of TtGalA is the first report of a quaternary structure as a hexameric assembly in the α-galactosidase family.

Evidence of Diradicals Involved in the Yeast Transketolase Catalyzed Keto-Transferring Reactions.

Transketolase (TK) catalyzes a reversible transfer of a two-carbon (C2 ) unit between phosphoketose donors and phosphoaldose acceptors, for which the group-transfer reaction that follows a one- or two-electron mechanism and the force that breaks the C2"-C3" bond of the ketose donors remain unresolved. Herein, we report ultrahigh-resolution crystal structures of a TK (TKps) from Pichia stipitis in previously undiscovered intermediate states and support a diradical mechanism for a reversible group-transfer reaction. In conjunction with MS, NMR spectroscopy, EPR and computational analyses, it is concluded that the enzyme-catalyzed non-Kekulé diradical cofactor brings about the C2"-C3" bond cleavage/formation for the C2 -unit transfer reaction, for which suppression of activation energy and activation and destabilization of enzymatic intermediates are facilitated.

Evolution of archaeal Rib7 and eubacterial RibG reductases in riboflavin biosynthesis: Substrate specificity and cofactor preference.

Archaeal/fungal Rib7 and eubacterial RibG possess a reductase domain for ribosyl reduction in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for an amino and a carbonyl group of the pyrimidine ring, respectively. Here, several crystal structures of Methanosarcina mazei Rib7 are reported at 2.27-1.95 Šresolution, which are the first archaeal dimeric Rib7 structures. Mutational analysis displayed that no detectable activity was observed for the Bacillus subtilis RibG K151A, K151D, and K151E mutants, and the M. mazei Rib7 D33N, D33K, and E156Q variants, while 0.1-0.6% of the activity was detected for the M. mazei Rib7 N9A, S29A, D33A, and D57N variants. Our results suggest that Lys151 in B. subtilis RibG, while Asp33 together with Arg36 in M. mazei Rib7, ensure the specific substrate recognition. Unexpectedly, an endogenous NADPH cofactor is observed in M. mazei Rib7, in which the 2'-phosphate group interacts with Ser88, and Arg91. Replacement of Ser88 with glutamate eliminates the endogenous NADPH binding and switches preference to NADH. The lower melting temperature of ∼10 °C for the S88E and R91A mutants suggests that nature had evolved a tightly bound NADPH to greatly enhance the structural stability of archaeal Rib7.

Crystal structures of Staphylococcal SaeR reveal possible DNA-binding modes.

Two-component system SaeRS of Staphylococcus regulates virulence factor expression through phosphorylation of the DNA-binding regulator SaeR by the sensor histidine kinase SaeS. Here crystal structures of the DNA-binding domain (DBD) of SaeR from two Staphylococcal species Staphylococcus epidermidis and Staphylococcus aureus were determined and showed similar folds. Analyzing the DNA binding activity of three mutants of SeSaeR, we observed that Thr217 is important in binding to the phosphate group of DNA and Trp219 may interact with the base pairs. Additionally, the tandem arrangement of DBD may represent a possible way for SaeR oligomerization on DNA.

SH3-like motif-containing C-terminal domain of staphylococcal teichoic acid transporter suggests possible function.

The negatively charged bacterial polysaccharides-wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising a transmembrane subunit TagG and an ATPase subunit TagH. We determined the crystal structure of the C-terminal domain of TagH (TagH-C) to investigate its function. The structure shows an N-terminal SH3-like subdomain wrapped by a C-terminal subdomain with an anti-parallel ß-sheet and an outer shell of α-helices. A stretch of positively charged surface across the subdomain interface is flanked by two negatively charged regions, suggesting a potential binding site for negatively charged polymers, such as WTAs or acidic peptide chains. Proteins 2016; 84:1328-1332. © 2016 Wiley Periodicals, Inc.

Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis.

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) plays a key role in sialic acid production. It is different from the non-hydrolyzing enzymes for bacterial cell wall biosynthesis, and it is feed-back inhibited by the downstream product CMP-Neu5Ac. Here the complex crystal structure of the N-terminal epimerase part of human GNE shows a tetramer in which UDP binds to the active site and CMP-Neu5Ac binds to the dimer-dimer interface. The enzyme is locked in a tightly closed conformation. By comparing the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, we propose a possible explanation for the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. On the other hand, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria. Moreover, full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme.

Structure and mechanism of an antibiotics-synthesizing 3-hydroxykynurenine C-methyltransferase.

Streptosporangium sibiricum SibL catalyzes the methyl transfer from S-adenosylmethionine (SAM) to 3-hydroxykynurenine (3-HK) to produce S-adenosylhomocysteine (SAH) and 3-hydroxy-4-methyl-kynurenine for sibiromycin biosynthesis. Here, we present the crystal structures of apo-form Ss-SibL, Ss-SibL/SAH binary complex and Ss-SibL/SAH/3-HK ternary complex. Ss-SibL is a homodimer. Each subunit comprises a helical N-terminal domain and a Rossmann-fold C-terminal domain. SAM (or SAH) binding alone results in domain movements, suggesting a two-step catalytic cycle. Analyses of the enzyme-ligand interactions and further mutant studies support a mechanism in which Tyr134 serves as the principal base in the transferase reaction of methyl group from SAM to 3-HK.

Crystal structure of a conserved hypothetical protein MJ0927 from Methanocaldococcus jannaschii reveals a novel quaternary assembly in the Nif3 family.

A Nif3 family protein of Methanocaldococcus jannaschii, MJ0927, is highly conserved from bacteria to humans. Although several structures of bacterial Nif3 proteins are known, no structure representing archaeal Nif3 has yet been reported. The crystal structure of Methanocaldococcus jannaschii MJ0927 was determined at 2.47 Å resolution to understand the structural differences between the bacterial and archaeal Nif3 proteins. Intriguingly, MJ0927 is found to adopt an unusual assembly comprising a trimer of dimers that forms a cage-like architecture. Electrophoretic mobility-shift assays indicate that MJ0927 binds to both single-stranded and double-stranded DNA. Structural analysis of MJ0927 reveals a positively charged region that can potentially explain its DNA-binding capability. Taken together, these data suggest that MJ0927 adopts a novel quartenary architecture that could play various DNA-binding roles in Methanocaldococcus jannaschii.

Crystal structure of Deinococcus radiodurans RecQ helicase catalytic core domain: the interdomain flexibility.

RecQ DNA helicases are key enzymes in the maintenance of genome integrity, and they have functions in DNA replication, recombination, and repair. In contrast to most RecQs, RecQ from Deinococcus radiodurans (DrRecQ) possesses an unusual domain architecture that is crucial for its remarkable ability to repair DNA. Here, we determined the crystal structures of the DrRecQ helicase catalytic core and its ADP-bound form, revealing interdomain flexibility in its first RecA-like and winged-helix (WH) domains. Additionally, the WH domain of DrRecQ is positioned in a different orientation from that of the E. coli RecQ (EcRecQ). These results suggest that the orientation of the protein during DNA-binding is significantly different when comparing DrRecQ and EcRecQ.

Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element.

The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the solution and crystal structures of the free kpRstA RD, DBD and DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion. Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein-DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.

Crystal structures of the archaeal UDP-GlcNAc 2-epimerase from Methanocaldococcus jannaschii reveal a conformational change induced by UDP-GlcNAc.

Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) 2-epimerase catalyzes the interconversion of UDP-GlcNAc to UDP-N-acetylmannosamine (UDP-ManNAc), which is used in the biosynthesis of cell surface polysaccharides in bacteria. Biochemical experiments have demonstrated that mutation of this enzyme causes changes in cell morphology and the thermoresistance of the cell wall. Here, we present the crystal structures of Methanocaldococcus jannaschii UDP-GlcNAc 2-epimerase in open and closed conformations. A comparison of these crystal structures shows that upon UDP and UDP-GlcNAc binding, the enzyme undergoes conformational changes involving a rigid-body movement of the C-terminal domain. We also present the crystal structure of Bacillus subtilis UDP-GlcNAc 2-epimerase in the closed conformation in the presence of UDP and UDP-GlcNAc. Although a structural overlay of these two closed-form structures reveals that the substrate-binding site is evolutionarily conserved, some areas of the allosteric site are distinct between the archaeal and bacterial UDP-GlcNAc 2-epimerases. This is the first report on the crystal structure of archaeal UDP-GlcNAc 2-epimerase, and our results clearly demonstrate the changes between the open and closed conformations of this enzyme.

Crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the response regulator SaeR from Staphylococcus epidermidis.

SaeR is the response regulator of the SaeRS two-component signal transduction system, which is involved in regulating bacterial autolysis and biofilm formation. SaeR comprises an N-terminal receiver domain and a C-terminal effector domain. The effector domain possesses DNA-binding and transactivation functions. Here, the effector domain of SaeR from Staphylococcus epidermidis was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.15 Å and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.20, b = 53.78, c = 111.66 Å. Determining the structure will provide insights into the mechanisms underlying DNA binding.

Evolution of vitamin B2 biosynthesis: eubacterial RibG and fungal Rib2 deaminases.

Eubacterial RibG and yeast Rib2 possess a deaminase domain for pyrimidine deamination in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for ribose and ribitol, respectively. Here, the crystal structure of Bacillus subtilis RibG in complex with a deaminase product is reported at 2.56 Å resolution. Two loops move towards the product on substrate binding, resulting in interactions with the ribosyl and phosphate groups and significant conformational changes. The product carbonyl moiety is bent out of the pyrimidine ring to coordinate to the catalytic zinc ion. Such distortions in the bound substrate and product may play an essential role in enzyme catalysis. The yeast Rib2 structure was modelled and a mutational analysis was carried out in order to understand the mechanism of substrate recognition in these two enzymes. Detailed structural comparisons revealed that the two consecutive carbonyl backbones that occur prior to the PCXXC signature constitute a binding hole for the target amino group of the substrate. This amino-binding hole is essential in B. subtilis RibG and is also conserved in the RNA/DNA-editing deaminases.

Crystallization and preliminary X-ray diffraction analysis of the Nif3-family protein MJ0927 from Methanocaldococcus jannaschii.

MJ0927 is a member of the Nif3 family and is widely distributed across living organisms. Although several crystal structures of Nif3 proteins have been reported, structural information on archaeal Nif3 is still limited. To understand the structural differences between bacterial and archaeal Nif3 proteins, MJ0927 from Methanocaldococcus jannaschii was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.47 Šand belonged to the orthorhombic space group C222, with unit-cell parameters a = 81.21, b = 172.94, c = 147.42 Å. Determination of this structure may provide insights into the function of MJ0927.

(1)H, (13)C and (15)N resonance assignments of the C-terminal DNA-binding domain of RstA protein from Klebsiella pneumoniae.

Bacterial cells often use two-component signal transduction systems to regulate genes in response to environmental stimuli. The RstA/RstB system is a two-component regulatory system consisting of the membrane sensor, RstB, and its cognate response regulator RstA. The RstA of Klebsiella pneumoniae consists of a N-terminal receiver domain (NRD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of the response regulator induces a conformational change in the regulatory domain of RstA, which results in activation of the effector domain to regulate the downstream genes, including the ferrous iron transport system (Feo), at low-pH condition. Here we report the (1)H, (13)C and (15)N resonance assignments and secondary structure identification of the DBD of RstA from K. pneumoniae as a first step for unraveling the structural and functional relationship of the RstA/RstB two component system.

Expression, purification, crystallization and preliminary X-ray analysis of ribitol-5-phosphate cytidylyltransferase from Bacillus subtilis.

TarI is a ribitol-5-phosphate cytidylyltransferase that catalyzes the formation of CDP-ribitol, which is involved in the biosynthesis of wall teichoic acids, from CTP and ribitol 5-phosphate. TarI from Bacillus subtilis (BsTarI) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.78 Šand belonged to the monoclinic space group C2, with unit-cell parameters a = 103.74, b = 60.97, c = 91.80 Å, ß = 113.48°. The initial structural model indicated that the crystals of BsTarI contained a dimer in the asymmetric unit.