Structures of the NDP-pyranose mutase belonging to glycosyltransferase family 75 reveal residues important for Mn2+ coordination and substrate binding.

Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDP-pyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear. Here we report the first crystal structures of GT75 family NDP-pyranose mutases. The novel structures of GT75 member MtdL in complex with Mn2+ and GDP, GDP-D-glucopyranose, GDP-L-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn2+ coordination, substrate binding, and catalytic reactions. We also provide a possible catalytic mechanism for this unique type of NDP-pyranose mutase. Taken together, our results highlight key elements of an enzyme family important for furanose biosynthesis.

Gongronella sp. w5 hydrolyzes plant sucrose and releases fructose to recruit phosphate-solubilizing bacteria to provide plants with phosphorus.

The mechanisms of how plant-beneficial rhizospheric fungi interact with the soil microbial community to promote plant growth by facilitating their phosphorus acquisition are poorly understood. This work supported that a Mucoromycotina fungus, Gongronella sp. w5 (w5), could promote phosphorus uptake of Medicago truncatula by increasing the available phosphorus (P) in the soil. The abundance of phosphate-solubilizing bacteria (PSB) and the activity of alkaline phosphatase (ALP) in alfalfa rhizosphere soil increased after w5 inoculation. Further analysis showed that w5 donated a portion of ALP activity and also stimulated the PSB to secrete ALP during plant-w5-PSB interaction to help release more available P in the rhizosphere of M. truncatula. Unlike most plant-beneficial rhizospheric fungi that mainly acquire hexoses from plants, w5 gained sucrose directly from the host plant and then recruited PSB to aid P acquisition by hydrolyzing sucrose and releasing mainly fructose to induce PSB to secrete ALP. IMPORTANCE: This work supported that after absorbing plant sucrose, Gongronella sp. w5 mainly releases sucrose hydrolysis product fructose into the environment. Fructose was used as a carbon source and signaling molecules to induce PSB to co-produce higher alkaline phosphatase activity, releasing soil-available phosphorus and promoting M. truncatula growth. This is the first report that plant-beneficial fungi could directly metabolize sucrose from plants and then recruit PSB to aid P acquisition by providing fructose. Our findings revealed the diversity in pathways of plant-fungi-PSB interactions on soil P acquisition and deepened our understanding of the cooperation of growth-promoting microorganisms in plant rhizosphere.

Two Zn2Cys6-type transcription factors respond to aromatic compounds and regulate the expression of laccases in the white-rot fungus Trametes hirsuta.

White-rot fungi differentially express laccases when they encounter aromatic compounds. However, the underlying mechanisms are still being explored. Here, proteomics analysis revealed that in addition to increased laccase activity, proteins involved in sphingolipid metabolism and toluene degradation as well as some cytochrome P450s (CYP450s) were differentially expressed and significantly enriched during 48 h of o-toluidine exposure, in Trametes hirsuta AH28-2. Two Zn2Cys6-type transcription factors (TFs), TH8421 and TH4300, were upregulated. Bioinformatics docking and isothermal titration calorimetry assays showed that each of them could bind directly to o-toluidine and another aromatic monomer, guaiacol. Binding to aromatic compounds promoted the formation of TH8421/TH4300 heterodimers. TH8421 and TH4300 silencing in T. hirsuta AH28-2 led to decreased transcriptional levels and activities of LacA and LacB upon o-toluidine and guaiacol exposure. EMSA and ChIP-qPCR analysis further showed that TH8421 and TH4300 bound directly with the promoter regions of lacA and lacB containing CGG or CCG motifs. Furthermore, the two TFs were involved in direct and positive regulation of the transcription of some CYP450s. Together, TH8421 and TH4300, two key regulators found in T. hirsuta AH28-2, function as heterodimers to simultaneously trigger the expression of downstream laccases and intracellular enzymes. Monomeric aromatic compounds act as ligands to promote heterodimer formation and enhance the transcriptional activities of the two TFs.IMPORTANCEWhite-rot fungi differentially express laccase isoenzymes when exposed to aromatic compounds. Clarification of the molecular mechanisms underlying differential laccase expression is essential to elucidate how white-rot fungi respond to the environment. Our study shows that two Zn2Cys6-type transcription factors form heterodimers, interact with the promoters of laccase genes, and positively regulate laccase transcription in Trametes hirsuta AH28-2. Aromatic monomer addition induces faster heterodimer formation and rate of activity. These findings not only identify two new transcription factors involved in fungal laccase transcription but also deepen our understanding of the mechanisms underlying the response to aromatics exposure in white-rot fungi.

An apoptosis-inducing factor controls programmed cell death and laccase expression during fungal interactions.

Apoptotic-like programmed cell death (PCD) is one of the main strategies for fungi to resist environmental stresses and maintain homeostasis. The apoptosis-inducing factor (AIF) has been shown in different fungi to trigger PCD through upregulating reactive oxygen species (ROS). This study identified a mitochondrial localized AIF homolog, CcAIF1, from Coprinopsis cinerea monokaryon Okayama 7. Heterologous overexpression of CcAIF1 in Saccharomyces cerevisiae caused apoptotic-like PCD of the yeast cells. Ccaif1 was increased in transcription when C. cinerea interacted with Gongronella sp. w5, accompanied by typical apoptotic-like PCD in C. cinerea, including phosphatidylserine externalization and DNA fragmentation. Decreased mycelial ROS levels were observed in Ccaif1 silenced C. cinerea transformants during cocultivation, as well as reduction of the apoptotic levels, mycelial growth, and asexual sporulation. By comparison, Ccaif1 overexpression led to the opposite phenotypes. Moreover, the transcription and expression levels of laccase Lcc9 decreased by Ccaif1 silencing but increased firmly in Ccaif1 overexpression C. cinerea transformants in coculture. Thus, in conjunction with our previous report that intracellular ROS act as signal molecules to stimulate defense responses, we conclude that CcAIF1 is a regulator of ROS to promote apoptotic-like PCD and laccase expression in fungal-fungal interactions. In an axenic culture of C. cinerea, CcAIF1 overexpression and H2O2 stimulation together increased laccase secretion with multiplied production yield. The expression of two other normally silent isozymes, Lcc8 and Lcc13, was unexpectedly triggered along with Lcc9. KEY POINTS: • Mitochondrial CcAIF1 induces PCD during fungal-fungal interactions • CcAIF1 is a regulator of ROS to trigger the expression of Lcc9 for defense • CcAIF1 overexpression and H2O2 stimulation dramatically increase laccase production.

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Sphingomicrobium clamense sp. nov., Isolated from Sediment of Clam Island Beach in China.

A Gram-stain-negative, non-flagellated, aerobic, ovoid or rod-shaped bacterium with motility, designated B8T, was isolated from the sediment of Clam Island beach, Liaoning province, China. The optimum growth of strain B8T occurred at 35 oC, pH 7.0, and in the presence of 4.0-5.0% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B8T formed a distinct lineage within the genus Sphingomicrobium and was closely related to Sphingomicrobium nitratireducens O-35T (98.3% sequence similarity), Sphingomicrobium aestuariivivum KCTC 42286T (96.9%), and Sphingomicrobium astaxanthinifaciens JCM 18551T (96.5%). The digital DNA-DNA hybridization and average nucleotide identity values between strain B8T and closely related strains were lower than 21.0% and 78.0%, much lower than the cutoff values of 70.0% and 95.0%, respectively, for bacterial species delineation. The dominant respiratory quinone of strain B8T was ubiquinone-10. The major fatty acids were Sum In Feature 8 (C18:1ω7c and/or C18:1ω6c), Sum In Feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C17:1ω6c, C18:1 2-OH, and C16:0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, glycolipids, and four unknown polar lipids. The DNA G + C content of strain B8T was 63.9%. Based on the phenotypic, phylogenetic, and chemotaxonomic analyses, strain B8T is considered a new species of Sphingomicrobium, for which the name Sphingomicrobium clamense sp. nov. is proposed. The type strain is B8T (= CGMCC 1.19486T = KCTC 92052T).

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization.

BACKGROUND: Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value. RESULTS: In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. CONCLUSIONS: This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

Maribacter aquimaris sp. nov., isolated from seawater adjacent to Fildes Peninsula, Antarctica.

A novel Gram-stain-negative, aerobic, and rod-shaped bacterium with gliding motility, named strain ANRC-HE7T, was isolated from the seawater of Biological Bay adjacent to Fildes Peninsula, Antarctica. The optimal growth of this strain occurred at 28 °C, pH 7.5, and in the presence of 1.0% (w/v) NaCl. Strain ANRC-HE7T can produce amylase and harbors gene clusters involved in cellulose degradation. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain ANRC-HE7T formed a distinct lineage within the genus Maribacter and was closely related to Maribacter luteus RZ05T (98.4% sequence similarity), Maribacter polysiphoniae LMG 23671T (98.3%), and Maribacter arenosus CAU 1321T (97.3%). However, digital DNA-DNA hybridization and average nucleotide identity values between strain ANRC-HE7T and closely related strains were 17.4-49.1% and 70.9-92.7%, much lower than the cutoff values of 70% and 95%, respectively. On the other hand, strain ANRC-HE7T shared characteristics with most type strains within the genus. Its respiratory quinone was MK-6. The major fatty acids were iso-C15:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), and anteiso-C15:0. The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids, four unidentified phospholipids, and five unidentified glycolipids. The DNA G + C content of strain ANRC-HE7T was 40.1%. Based on the results of the biochemical, phylogenetic, and chemotaxonomic analyses, strain ANRC-HE7T is suggested to represent a novel species of the genus Maribacter, for which the name Maribacter aquimaris sp. nov. is proposed. The type strain is ANRC-HE7T (= MCCC 1K03787T = KCTC 72532T).

ThhspA1 is involved in lacA transcriptional regulation of Trametes hirsuta AH28-2 exposed to o-toluidine.

White rot fungi, especially Trametes spp., respond to a wide range of aromatic compounds and dramatically enhance laccase activity, while the activation mechanisms remain to be elucidated. Here, we show that an Hsp70 homolog named ThhspA1 regulates the transcription of laccase LacA in Trametes hirsuta AH28-2 when confronted with o-toluidine. ThhspA1 is pulled down by lacA promoter sequence from the nuclear mixture extracted from T. hirsuta AH28-2 induced by 2 mM o-toluidine. Silencing of ThhspA1 results in a sharp decrease in lacA transcripts and laccase activity in vivo. By comparison, ThhspA1 overexpression does not affect lacA transcription, and laccase activity shows slight enhancement or remains unchanged upon induction with o-toluidine. Electrophoretic mobility shift assays suggest a direct interaction between ThhspA1 and the lacA promoter region. Further investigation shows that the integrity of ThhspA1 is critical since its substrate binding domain (SBD) and nucleotide-binding domain (NBD) are both necessary for DNA binding, with a higher affinity of SBD than NBD based on fluorescence polarization assay. Our results demonstrate that ThhspA1 functions as an aromatic-stress-related DNA binding transcriptional factor required for LacA expression.

Coprinopsis cinerea Uses Laccase Lcc9 as a Defense Strategy To Eliminate Oxidative Stress during Fungal-Fungal Interactions.

Frequently, laccases are triggered during fungal cocultivation for overexpression. The function of these activated laccases during coculture has not been clarified. Previously, we reported that Gongronella sp. w5 (w5) (Mucoromycota, Mucoromycetes) specifically triggered the laccase Lcc9 overexpression in Coprinopsis cinerea (Basidiomycota, Agaricomycetes). To systematically analyze the function of the overexpressed laccase during fungal interaction, C. cinerea mycelia before and after the initial Lcc9 overexpression were chosen for transcriptome analysis. Results showed that accompanied by specific utilization of fructose as carbohydrate substrate, oxidative stress derived from antagonistic compounds secreted by w5 appears to be a signal critical for laccase production in C. cinerea. A decrease in reactive oxygen species (ROS) in the C. cinerea wild-type strain followed the increase in laccase production, and then lcc9 transcription and laccase activity stopped. By comparison, increased H2O2 content and mycelial ROS levels were observed during the entire cocultivation in lcc9 silenced C. cinerea strains. Moreover, lcc9 silencing slowed down the C. cinerea mycelial growth, affected hyphal morphology, and decreased the asexual sporulation in coculture. Our results showed that intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Lcc9 takes part in a defense strategy to eliminate oxidative stress during the interspecific interaction with w5. IMPORTANCE The overproduction of laccase during interspecific fungal interactions is well known. However, the exact role of the upregulated laccases remains underexplored. Based on comparative transcriptomic analysis of C. cinerea and gene silencing of laccase Lcc9, here we show that oxidative stress derived from antagonistic compounds secreted by Gongronella sp. w5 was a signal critical for laccase Lcc9 production in Coprinopsis cinerea. Intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Ultimately, Lcc9 takes part in a defense strategy to eliminate oxidative stress and help cell growth and development during the interspecific interaction with Gongronella sp. w5. These findings deepened our understanding of fungal interactions in their natural population and communities.

A Superefficient Ochratoxin A Hydrolase with Promising Potential for Industrial Applications.

As the most seriously controlled mycotoxin produced by Aspergillus spp. and Penicillium spp., ochratoxin A (OTA) results in various toxicological effects and widely contaminates agro-products. Biological detoxification is the highest priority regarding OTA in food and feed industry, but currently available detoxification enzymes have relatively low effectiveness in terms of time and cost. Here we show a superefficient enzyme, ADH3, identified from Stenotrophomonas acidaminiphila that has a strong ability to transform OTA into nontoxic ochratoxin-α by acting as an amidohydrolase. Recombinant ADH3 (1.2 µg/mL) completely degrades 50 µg/L OTA within 90 s, while the other most efficient OTA hydrolases available take several hours. The kinetic constant showed that rADH3 (Kcat/Km) catalytic efficiency was 56.7 to 35,000 times higher than those of previous hydrolases rAfOTase, rOTase, and commercial carboxypeptidase A (CPA). Protein structure-based assay suggested that ADH3 has a preference for hydrophobic residues to form a larger hydrophobic area than other detoxifying enzymes at the cavity of the catalytic sites, and this structure allows OTA easier access to the catalytic sites. In addition, ADH3 shows considerable temperature adaptability to exert hydrolytic function at the temperature down to 0°C or up to 70°C. Collectively, we report a superefficient OTA detoxifying enzyme with promising potential for industrial applications. IMPORTANCE Ochratoxin A (OTA) can result in various toxicological effects and widely contaminates agro-products and feedstuffs. OTA detoxifications by microbial strains and bio-enzymes are significant to food safety. Although previous studies showed OTA could be transformed through several pathways, the ochratoxin-α pathway is recognized as the most effective one. However, the most currently available enzymes are not efficient enough. Here, a superefficient hydrolase, ADH3, which can completely transform 50 µg/L OTA into ochratoxin-α within 90 s was screened and characterized. The hydrolase ADH3 shows considerable temperature adaptability (0 to 70°C) to exert the hydrolytic function. Findings of this study supplied an efficient OTA detoxifying enzyme and predicted the superefficient degradation mechanism, laying a foundation for future industrial applications.

Enhanced extracellular raw starch-degrading α-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization.

BACKGROUND: A raw starch-degrading α-amylase from Pontibacillus sp. ZY (AmyZ1), previously screened by our laboratory, showed a promising application potential for starch-processing industries. However, the AmyZ1 secretory production still under investigation, which seriously restricts its application in the starch-processing industry. On the other hand, Bacillus subtilis is widely used to achieve the extracellular expression of target proteins. RESULTS: AmyZ1 secretory production was achieved in B. subtilis and was enhanced by promoter engineering and translation initiation efficiency optimization. First, based on the different phase-dependent promoters, the dual-promoter PspoVG-PspoVG142 was constructed by combining dual-promoter engineering and promoter modification. The corresponding strain BZd34 showed an extracellular AmyZ1 activity of 1437.6 U/mL during shake flask cultivation, which was 3.11-fold higher than that of the original strain BZ1 (PgroE). Then, based on translation initiation efficiency optimization, the best strain BZd343 containing optimized 5'-proximal coding sequence (opt3) produced the highest extracellular α-amylase activity of 1691.1 U/mL, which was 3.65-fold higher than that of the strain BZ1. Finally, cultivation of BZd343 in 3-L fermenter exhibited an extracellular AmyZ1 activity of 14,012 U/mL at 48 h, with productivity of 291.9 U/mL·h. CONCLUSIONS: This is the first report of recombinant expression of AmyZ1 in B. subtilis and the expression level of AmyZ1 represents the highest raw starch-degrading α-amylase level in B. subtilis to date. The high-level expression of AmyZ1 in this work provides a foundation for its industrial production. The strategies used in this study also provide a strategic reference for improving the secretory expression of other enzymes in B. subtilis.

Sedimentimonas flavescens gen. nov., sp. nov., isolated from sediment of Clam Island, Liaoning Province.

A novel Gram-stain negative, aerobic and ovoid to short rod shaped bacterium with a single polar flagellum, named strain B57T, was isolated from sediment of Clam Island, Liaoning Province, China. The optimal growth of this strain was found to occur at 37 °C, pH 6-6.5, and in the presence of 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B57T forms a distinct lineage within the family Rhodobacteraceae, sharing high 16S rRNA gene sequence similarity with Sinirhodobacter populi sk2b1T (97.4%). The average amino acid identity of B57T and the closely related species were lower than the threshold level for genus delineation. The dominant respiratory quinone of strain B57T was identified as Q-10. The major fatty acids were found to be Summed Feature 8 (C18:1ω7c and/or C18:1ω6c), Summed Feature 3 (C16:1ω7c and/or C16:1ω6c) and C16: 0. The polar lipids were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, diphosphatidylglycerol, two unidentified phospholipids, one unidentified glycolipid, and one unidentified lipid. The DNA G + C content of strain B57T was determined to be 64.1 mol%. Based on the biochemical, phylogenetic and chemotaxonomic analysis, strain B57T is concluded to represent a novel species of a novel genus, for which the name Sedimentimonas flavescens gen. nov., sp. nov.is proposed. The type strain is B57T (= CGMCC1.19488T = KCTC 92053T).

Comparison of performances of different fungal laccases in delignification and detoxification of alkali-pretreated corncob for bioethanol production.

The performance of the alkaline fungal laccase PIE5 (pH 8.5) in the delignification and detoxification of alkali-pretreated corncob to produce bioethanol was evaluated and compared with that of the neutral counterpart (rLcc9, 6.5), with the acidic laccase rLacA (4.0) was used as an independent control. Treatment with the three laccases facilitated bioethanol production compared with their respective controls. The lignin contents of alkali-pretreated corncob reduced from 4.06%, 5.06%, and 7.80% to 3.44%, 3.95%, and 5.03%, after PIE5, rLcc9, and rLacA treatment, respectively. However, the performances of the laccases were in the order rLacA > rLcc9 > PIE5 in terms of decreasing total phenol concentration (0.18, 0.36, and 0.67 g/l), boosting ethanol concentration (8.02, 7.51, and 7.31 g/l), and volumetric ethanol productivity (1.34, 0.94, and 0.91 g/l hr), and shortening overall fermentation time. Our results would inform future attempts to improve laccases for ethanol production. Furthermore, based on our data and the fact that additional procedures, such as pH adjustment, are needed during neutral/alkaline fungal laccase treatment, we suggest acidic fungal laccases may be a better choice than neutral/alkaline fungal laccases in bioethanol production.

Mechanism of the salt activation of laccase Lac15.

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize various substrates, and those which are tolerant to and even activated by salts have attracted a lot of attention due to their application potential in certain industries. The mechanism of the salt activation of laccases is awaiting to be elucidated yet. Our previous study (Li, Xie et al. 2018) supposed that the salt activation of marine laccase Lac15 might be attributed to Cl- ion specifically binding to some local sites to interfere substrate binding and/or electron transfer. In this study, we found two sites whose mutations resulted in elimination of the salt activation of Lac15's activity towards catechol and dopamine respectively, and revealed that the mutations affected the activity by altering both Em and kcat, demonstrating the supposed mechanism. A model for the salt activation of laccases was accordingly proposed, albeit some details are to be elucidated.

An unusual GH1 ß-glucosidase from marine sediment with ß-galactosidase and transglycosidation activities for superior galacto-oligosaccharide synthesis.

A novel ß-glucosidase, BglD1 with high ß-galactosidase and transglycosidation activities, was screened and cloned from the deep-sea bacterium Bacillus sp. D1. BglD1 exhibited the maximal ß-glucosidase and ß-galactosidase activities at 55-60 °C and pH 5.5-6.0. The enzyme maintained approximately 50% of its original activity at 35 °C and pH 6.0 after 120-h incubation. When applied to synthesize galacto-oligosaccharides (GOS), BglD1 generated 118.3 g/L GOS (33.8% (w/w)) from 350 g/L lactose, with trisaccharide Gal-ß(1 → 3)-Lac and disaccharide Gal-ß(1 → 4)-Gal as the main components. Furthermore, BglD1 could hydrolyze lactose in milk and produce GOS simultaneously. Using milk as the substrate, BglD1 hydrolyzed 88.5% lactose and produced 3.3 g/L GOS after incubation at 30 °C for 1 h. To improve the transglycosidation activity, a mutant BglD1:E224T was generated based on the semi-rational design. The GOS yield of BglD1:E224T was 11.5% higher than that of BglD1 when using lactose solution as the substrate. Thus, BglD1 and the mutant could be used as beneficial alternatives of the existing ß-galactosidases for the production of GOS.

Structural basis for histone H3K4me3 recognition by the N-terminal domain of the PHD finger protein Spp1.

Saccharomyces cerevisiae Spp1, a plant homeodomain (PHD) finger containing protein, is a critical subunit of the histone H3K4 methyltransferase complex of proteins associated with Set1 (COMPASS). The chromatin binding affinity of the PHD finger of Spp1 has been proposed to modulate COMPASS activity. During meiosis, Spp1 plays another role in promoting programmed double-strand break (DSB) formation by binding H3K4me3 via its PHD finger and interacting with a DSB protein, Mer2. However, how the Spp1 PHD finger performs site-specific readout of H3K4me3 is still not fully understood. In the present study, we determined the crystal structure of the highly conserved Spp1 N-terminal domain (Sc_Spp1NTD) in complex with the H3K4me3 peptide. The structure shows that Sc_Spp1NTD comprises a PHD finger responsible for methylated H3K4 recognition and a C3H-type zinc finger necessary to ensure the overall structural stability. Our isothermal titration calorimetry results show that binding of H3K4me3 to Sc_Spp1NTD is mildly inhibited by H3R2 methylation, weakened by H3T6 phosphorylation, and abrogated by H3T3 phosphorylation. This histone modification cross-talk, which is conserved in the Saccharomyces pombe and mammalian orthologs of Sc_Spp1 in vitro, can be rationalized structurally and might contribute to the roles of Spp1 in COMPASS activity regulation and meiotic recombination.

Improving the thermostability of a GH97 dextran glucosidase by rational design.

This study was aimed at improving the thermostability of dextran glucosidase PspAG97A, a member of the glycoside hydrolase family 97, from Pseudoalteromonas sp. K8. A total of 9 lysine residues were chosen using the TKSA-MC program based on the optimization of surface charge-charge interactions and were mutated to glutamate for shifting the enzyme's isoelectric point off its optimum pH value. Three mutants K75E, K363E and K420E showed enhanced thermostability. The triple mutant, K75E/K363E/K420E, was found to be the best with a 7.3-fold increase in half-life (t1/2) at 33 °C compared to that of the wild-type (WT). Most importantly, this mutant showed comparable enzymatic activity to that of the WT protein. Structural modelling demonstrated that increased surface charge-charge interactions and optimization of surface hydrophobic and electrostatic contacts contributed to the improved thermostability displayed by K75E/K363E/K420E.

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P.

A bacterial inverting glycosyltransferase EarP transfers rhamnose from dTDP-ß-l-rhamnose (TDP-Rha) to Arg32 of translation elongation factor P (EF-P) to activate its function. We report here the structural and biochemical characterization of Pseudomonas aeruginosa EarP. In contrast to recently reported Neisseria meningitidis EarP, P. aeruginosa EarP exhibits differential conformational changes upon TDP-Rha and EF-P binding. Sugar donor binding enhances acceptor binding to EarP, as revealed by structural comparison between the apo-, TDP-Rha-, and TDP/EF-P-bound forms and isothermal titration calorimetry experiments. In vitro EF-P rhamnosylation combined with active-site geometry indicates that Asp16 corresponding to Asp20 of N. meningitidis EarP is the catalytic base, whereas Glu272 is another putative catalytic residue. Our study should provide the basis for EarP-targeted inhibitor design against infections from P. aeruginosa and other clinically relevant species.IMPORTANCE Posttranslational rhamnosylation of EF-P plays a key role in Pseudomonas aeruginosa, establishing virulence and antibiotic resistance, as well as survival. The detailed structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP from P. aeruginosa not only demonstrates that sugar donor TDP-Rha binding enhances acceptor EF-P binding to EarP but also should provide valuable information for the structure-guided development of its inhibitors against infections from P. aeruginosa and other EarP-containing pathogens.

Statistical coupling analysis uncovers sites crucial for the proton transfer in laccase Lac15.

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize wide range of compounds thus have great application potential in diverse industries. The catalytic mechanisms of laccases have been extensively studied, while the details of proton transfer remain to be fully elucidated. In this study, we tried to uncover the sites that are crucial for the proton transfer of microbial laccase Lac15. A residue near the trinuclear copper center, D396, was indicated by statistical coupling analysis (SCA) and structural alignment to be an important site like D93, which is conserved in laccases and believed crucial for the catalysis by facilitating proton transfer. A representative mutant at this site, D396A, similar to D93A, exhibited significantly impaired catalysis with the global structure and substrate binding slightly perturbed. The mutation resulted in stay of the intermediate I, which would accept a proton to proceed to next catalysis stage, suggesting D396 might play a critical role in the proton transfer. Our finding may help to completely elucidate the proton transfer mechanism in laccases.