Molecular insights into substrate promiscuity of CotA laccase catalyzing lignin-phenol derivatives.

CotA laccases are multicopper oxidases known for promiscuously oxidizing a broad range of substrates. However, studying substrate promiscuity is limited by the complexity of electron transfer (ET) between substrates and laccases. Here, a systematic analysis of factors affecting ET including electron donor acceptor coupling (ΗDA), driving force (ΔG) and reorganization energy (λ) was done. Catalysis rates of syringic acid (SA), syringaldehyde (SAD) and acetosyringone (AS) (kcat(SAD) > kcat(SA) > kcat(AS)) are not entirely dependent on the ability to form phenol radicals indicated by ΔG and λ calculated by Density Functional Theory (SA < SAD ≈ AS). In determined CotA/SA and CotA/SAD structures, SA and SAD bound at 3.9 and 3.7 Å away from T1 Cu coordinating His419 ensuring a similar ΗDA. Abilities of substrate to form phenol radicals could mainly account for difference between kcat(SAD) and kcat(SA). Furthermore, substrate pocket is solvent exposed at the para site of substrate's phenol hydroxyl, which would destabilize binding of AS in the same orientation and position resulting in low kcat. Our results indicated shallow partially covered binding site with propensity of amino acids distribution might help CotA discriminate lignin-phenol derivatives. These findings give new insights for developing specific catalysts for industrial application.

Structure-based rational design of chitosanase CsnMY002 for high yields of chitobiose.

Chitosan oligosaccharides (COS) are attractive active molecules for biomedical applications. Currently, the prohibitively high cost of producing fully defined COS hampers extensive studies on their biological activity and restricts their use in various industries. Thus, cost-effective production of pure COS is of major importance. In this report, chitosanase from Bacillus subtilis MY002 (CsnMY002) was prepared for COS production. The structure of apo CsnMY002 displayed an unexpected tunnel-like substrate-binding site and the structure of the CsnMY002_E19A/(GlcN)6 complex highlighted the "4 + 2″ splitting of hexaglucosamine even though the "3 + 3″ splitting is also observed in the TLC analysis of the enzyme products for hexaglucosamine. Structure based rational design was performed to generate mutants for chitobiose production. The CsnMY002_G21 K mutant produced chitobiose with a relative content > 87 % from chitosan with a low degree of acetylation, and 50.65 mg chitobiose with a purity > 98 % was prepared from 100 mg chitosan. The results provide insight on the catalytic mechanism of chitosanase and underpin future biomedical applications of pure chitobiose.

Quantitation of nucleoprotein complexes by UV absorbance and Bradford assay.

Despite the importance of studying nucleoprotein complexes, no appropriate method for quantifying them is available. Here, a UV absorbance method using the formula "Cmg/mL = 1.55A280 - 0.76A260" were applied to quantify nucleoprotein complexes. After modification using two paired A260 and A280 values, the UV-derived formula-based method could accurately quantify proteins in nucleoprotein complexes. Otherwise, by taking the target protein as a standard, the Bradford assay can accurately quantify proteins in nucleoprotein complexes without interference by nucleic acids. The above methods were successfully applied to measure the concentration of MtuP49-CTG complexes of Mycobacterium tuberculosis. In conclusion, both the Bradford assay and the UV-derived formula-based method were appropriate for quantifying proteins in nucleoprotein complexes, which may make contributions to explore the interactions between proteins and nucleic acids at the molecular level.

Optimized methods for IL-17A refolding and anti-IL17A Fab production for co-crystallization with small molecules.

Refolding of human interleukin 17A (IL-17A) has been reported; however, the key refolding protocol was not robust enough to deliver consistent results and to be easily scaled up for crystallization. Here we report an optimized refolding method for IL-17A. Although co-crystal structures of IL-17A with ligands have been obtained with a high-affinity peptide and an anti-IL-17A Fab as stabilizers, neither the production yield nor the characterization of the IL-17A/Fab complex was reported. To facilitate co-crystallization of IL-17A with small-molecule compounds derived from our DNA encoded library, we also describe the method for yield enhancement of anti-IL-17A Fab production and characterize the IL-17A/Fab complex for the first time, providing an essential prerequisite for structure-based drug discovery targeting IL-17A.

Structural basis for monolignol oxidation by a maize laccase.

Plant laccases catalyse the oxidation of monolignols in lignification, a process reinforcing the cell wall of many different cell types that provide mechanical support, nutrient transportation and defence against pathogens in plants1. The isozymes display a broad range of substrate preferences. Here, the substrate preference of a laccase (ZmLac3) from Zea mays (maize) was characterized. The crystal structure of ZmLac3 revealed a compact and deep substrate-binding pocket, and the binding modes of sinapyl alcohol (SinA) and coniferyl alcohol (ConA) were solved. On the basis of structural data and kinetics analysis, we propose that the regionalization of polar and hydrophobic surfaces in the binding pocket of ZmLac3 is vital for defining the orientation of SinA/ConA binding. The extra methoxyl group in SinA makes substantial contributions to interactions between SinA and ZmLac3, which are absent in the ZmLac3-ConA complex. In summary, the polar and hydrophobic interactions between SinA/ConA and ZmLac3 determine the binding positions of the monolignols in ZmLac3. These results provide valuable insight about ZmLac3 catalysis and should aid industrial processes that use plant laccases.

Structure-guided engineering of ChKRED20 from Chryseobacterium sp. CA49 for asymmetric reduction of aryl ketoesters.

ChKRED20 is a robust NADH-dependent ketoreductase identified from the genome of Chryseobacterium sp. CA49 that can use 2-propanol as the ultimate reducing agent. The wild-type can reduce over 100 g/l ketones for some pharmaceutical relevant substrates, exhibiting a remarkable potential for industrial application. In this work, to overcome the limitation of ChKRED20 to aryl ketoesters, we first refined the X-ray crystal structure of ChKRED20/NAD+ complex at a resolution of 1.6 Å, and then performed three rounds of iterative saturation mutagenesis at critical amino acid sites to reshape the active cavity of the enzyme. For methyl 2-oxo-2-phenylacetate and ethyl 3-oxo-3-phenylpropanoate, several gain-of-activity mutants were achieved, and for ethyl 2-oxo-4-phenylbutanoate, improved mutants were achieved with kcat/Km increasing to 196-fold of the wild-type. All three substrates were completely reduced at 100 g/l loading catalyzed with selected ChKRED20 mutants, and deliver the corresponding chiral alcohols with >90% isolated yield and 97 - >99%ee.

Structural Insight into the Allosteric Coupling of Cu1 Site and Trinuclear Cu Cluster in CotA Laccase.

In laccase, type 1 copper (Cu1) was connected to the trinuclear copper center (TNC) by the conserved Cys-His bridge. An allosteric coupling between the two redox sites has been reported; however, the molecular mechanism underlining the allosteric coupling is unknown. In this study, ligands of the two type 3 copper sites, including His491 and His493, in CotA were mutated to Cys or Ala. The crystal structures revealed that mutations at His491 and His493 caused rearrangement of the hydrogen-bond network and geometric distortion of the TNC, which severely impaired the activities of mutants H493A, H493C, and H491C. In addition, the change in TNC affected hydrogen bonds around Cys492 in the mutants and led to Cu1 being partially reduced. These results not only decipher the mechanism of allosteric coupling between Cu1 and TNC in laccase, but also pave the way for laccase protein engineering.

Solution scattering study of the Bacillus subtilis PgdS enzyme involved in poly-γ-glutamic acids degradation.

The PgdS enzyme is a poly-γ-glutamic (γ-PGA) hydrolase, which has potential application for a controllable degradation of γ-PGA by enzymatic depolymerization; however, the structure of PgdS is still unknown. Here, to study in detail the full-length PgdS structure, we analyze the low-resolution architecture of PgdS hydrolase from Bacillus subtilis in solution using small angle X-ray scattering (SAXS) method. Combining with other methods, like dynamic light scattering and mutagenesis analyses, a model for the full length structure and the possible substrate delivery route of PgdS are proposed. The results will provide useful hints for future investigations into the mechanisms of γ-PGA degradation by the PgdS hydrolase and may provide valuable practical information.

Activity enhancement of CotA laccase by hydrophilic engineering, histidine tag optimization and static culture.

CotA protein from Bacillus subtilis is of laccase activity. The solubility of recombinant CotA is low, which hinders its application. In this study, histidine tag position optimization and hydrophilic engineering were applied to increase the yield and activity of CotA protein. The results showed that the protein yield of CotA with his tag at C-terminal (CH6-CotA) was four times of that of NH6-CotA (His tag at N-terminal). Then, 23 single mutants were constructed by substitutions of hydrophobic residues with hydrophilic amino acids. Among them, the protein yield of the mutant F207Y was increased by 30%; the catalytic activity (kcat/Km) of V403T and P455S was two and three times higher than that of CH6-CotA, respectively. Finally, triple mutant F2071Y/V403T/P455S with C-terminal his-tag (CH6-TSY) was constructed. When the proteins were expressed in microanaerobic condition, the activities of mutants CH6-P455S and CH6-TSY were enhanced about 48- and 42-folds compared to that of NH6-CotA in non-static culture.

Crystal structure of IspF from Bacillus subtilis and absence of protein complex assembly amongst IspD/IspE/IspF enzymes in the MEP pathway.

2-C-Methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF) is a key enzyme in the 2-C-Methyl-d-erythritol-4-phosphate (MEP) pathway of isoprenoid biosynthesis. This enzyme catalyzes the 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate (CDPME2P) to 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) with concomitant release of cytidine 5'-diphospate (CMP). Bacillus subtilis is a potential host cell for the production of isoprenoids, but few studies are performed on the key enzymes of MEP pathway in B. subtilis In this work, the high-resolution crystal structures of IspF in native and complex with CMP from B. subtilis have been determined. Structural comparisons indicate that there is a looser packing of the subunits of IspF in B. subtilis, whereas the solvent accessible surface of its active pockets is smaller than that in Escherichia coli. Meanwhile, the protein-protein associations of 2-C-Methyl-d-erythritol-4-phosphatecytidyltransferase (IspD), CDPME kinase (IspE) and IspF from B. subtilis and E. coli, which catalyze three consecutive steps in the MEP pathway, are analyzed by native gel shift and size exclusion chromatography methods. The data here show that protein complex assembly is not detectable. These results will be useful for isoprenoid biosynthesis by metabolic engineering.

[Advances in isoprene synthase research].

Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

Crystal structure and iterative saturation mutagenesis of ChKRED20 for expanded catalytic scope.

ChKRED20 is an efficient and robust anti-Prelog ketoreductase that can catalyze the reduction of ketones to chiral alcohols as pharmaceutical intermediates with great industrial potential. To overcome its limitation on the bioreduction of ortho-substituted acetophenone derivatives, the X-ray crystal structure of the apo-enzyme of ChKRED20 was determined at a resolution of 1.85 Å and applied to the molecular modeling and reshaping of the catalytic cavity via three rounds of iterative saturation mutagenesis together with alanine scanning and recombination. The mutant Mut3B was achieved with expanded catalytic scope that covered all the nine substrates tested as compared with two substrates for the wild type. It exhibited 13-20-fold elevated k cat/K m values relative to the wild type or to the first gain-of-activity mutant, while retaining excellent stereoselectivity toward seven of the substrates (98-> 99% ee). Another mutant 29G10 displayed complementary selectivity for eight of the ortho-substituted acetophenone derivatives, with six of them delivering excellent stereoselectivity (90-99% ee). Its k cat/K m value toward 1-(2-fluorophenyl)ethanone was 5.6-fold of the wild type. The application of Mut3B in elevated substrate concentrations of 50-100 g/l was demonstrated in 50-ml reactions, achieving 75-> 99% conversion and > 99% ee.

Structural Insight into the Specific DNA Template Binding to DnaG primase in Bacteria.

Bacterial primase initiates the repeated synthesis of short RNA primers that are extended by DNA polymerase to synthesize Okazaki fragments on the lagging strand at replication forks. It remains unclear how the enzyme recognizes specific initiation sites. In this study, the DnaG primase from Bacillus subtilis (BsuDnaG) was characterized and the crystal structure of the RNA polymerase domain (RPD) was determined. Structural comparisons revealed that the tethered zinc binding domain plays an important role in the interactions between primase and specific template sequence. Structural and biochemical data defined the ssDNA template binding surface as an L shape, and a model for the template ssDNA binding to primase is proposed. The flexibility of the DnaG primases from B. subtilis and G. stearothermophilus were compared, and the results implied that the intrinsic flexibility of the primase may facilitate the interactions between primase and various partners in the replisome. These results shed light on the mechanism by which DnaG recognizes the specific initiation site.

A structural and functional study on the 2-C-methyl-d-erythritol-4-phosphate cytidyltransferase (IspD) from Bacillus subtilis.

2-C-Methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) is an essential enzyme in the mevalonate-independent pathway of isoprenoid biosynthesis. This enzyme catalyzes 2-C-Methyl-d-erythritol 4-phosphate (MEP) and cytosine triphosphate (CTP) to 4-diphosphocytidyl-2-C-methyl-d-erythritol (CDPME) and inorganic pyrophosphate (PPi). Bacillus subtilis was a kind of excellent isoprene producer. However, the studies on the key enzymes of MEP pathway in B. subtilis were still absent. In this work, the crystal structures of IspD and IspD complexed with CTP from B.subtilis were determined. For the first time, the intact P-loop was observed in the apo structure of IspD enzyme. Structural comparisons revealed that the concerted movements of the P-loop and loops close to the active site were essential in the reaction catalyzed by IspD. Meanwhile, kinetic analysis showed that the CTP hydrolytic activity of IspD from B.subtilis was over two times higher than that from Escherichia coli. These results will be useful for future target-based screening of potential inhibitors and the metabolic engineering for isoprenoid biosynthesis.

Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site.

The CotA laccase from Bacillus subtilis is an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Šaway from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.

Structural insight into the oxidation of sinapic acid by CotA laccase.

Laccases can oxidize plenty of substrates by use of molecular oxygen as the final electron acceptor. The broad substrate spectrum is further expanded by using redox mediators in so-called laccase-mediator systems, but the structural studies on interactions between laccases and natural mediators are still absent. In this study, the crystal structure of CotA/sinapic acid complex is solved, structural comparison has revealed a novel substrate binding mode. The residue of His419 instead of His497 is bonding to the sinapic acid (SA) as the primary electron acceptor. Moreover, the binding of SA leads to 10° rotation on Arg416, our mutagenesis data exhibits that the residue Arg416 is crucial in the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and syringaldazine (SGZ). Furthermore, oxidation of several phenolic acids and one non-phenolic acid by CotA was investigated. By analyzing interactions between CotA and SA, it is indicated that the presence of methoxy groups in the ortho-position of the phenolic structure is crucial for the substrate recognition by CotA laccase. This work establishes structure-function relationships for laccase-natural mediator system.

A multiple-common-lines method to determine the orientation of snapshot diffraction patterns from single particles.

With the development of X-ray free-electron lasers (XFELs), it is possible to determine the three-dimensional structures of noncrystalline objects with coherent X-ray diffraction imaging. In this diffract-and-destroy mode, many snapshot diffraction patterns are obtained from the identical objects which are presented one by one in random orientations to the XFEL beam. Determination of the orientation of an individual object is essential for reconstruction of a three-dimensional structure. Here a new method, called the multiple-common-lines method, has been proposed to determine the orientations of high- and low-signal snapshot diffraction patterns. The mean errors of recovered orientations (α, ß, γ) of high- and low-signal patterns are about 0.14, 0.06, 0.12 and 0.77, 0.31, 0.60°, respectively; both sets of errors can meet the requirements of the reconstruction of a three-dimensional structure.