Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase.

Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.

The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para-hydroxybenzoate catabolism.

The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration, primarily during an enzymatic hydroxylation reaction. The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for 40 years. Here, we elucidate the molecular mechanism of the reaction in the conversion of para-hydroxybenzoate (PHB) to gentisate (GA, 2, 5-dihydroxybenzoate). Three genes (phgABC) from the PHB utilizer Brevibacillus laterosporus PHB-7a encode enzymes (p-hydroxybenzoyl-CoA ligase, p-hydroxybenzoyl-CoA hydroxylase and gentisyl-CoA thioesterase, respectively) catalyzing the conversion of PHB to GA via a route involving CoA thioester formation, hydroxylation concomitant with a 1, 2-shift of the acetyl CoA moiety and thioester hydrolysis. The shift of the carboxyl group was established rigorously by stable isotopic experiments with heterologously expressed phgABC, converting 2, 3, 5, 6-tetradeutero-PHB and [carboxyl-13 C]-PHB to 3, 4, 6-trideutero-GA and [carboxyl-13 C]-GA respectively. This is distinct from the NIH shifts of hydrogen and aceto substituents, where a single oxygenase catalyzes the reaction without the involvement of a thioester. The discovery of this three-step strategy for carboxyl group migration reveals a novel role of the CoA thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.

Toluene degradation via a unique metabolic route in indigenous bacterial species.

Tanneries are the primary source of toluene pollution in the environment and toluene due to its hazardous effects has been categorized as persistent organic pollutant. Present study was initiated to trace out metabolic fingerprints of three toluene-degrading bacteria isolated from tannery effluents of Southern Punjab. Using selective enrichment and serial dilution methods followed by biochemical, molecular and antibiotic resistance analysis, isolated bacteria were subjected to metabolomics analysis. GC-MS/LC-MS analysis of bacterial metabolites helped to identify toluene transformation products and underlying pathways. Three toluene-metabolizing bacteria identified as Bacillus paralicheniformis strain KJ-16 (IUBT4 and IUBT24) and Brevibacillus agri strain NBRC 15538 (IUBT19) were found tolerant to toluene and capable of degrading toluene. Toluene-degrading potential of these isolates was detected to be IUBT4 (10.35 ± 0.084 mg/h), IUBT19 (14.07 ± 3.14 mg/h) and IUBT24 (11.1 ± 0.282 mg/h). Results of GC-MS analysis revealed that biotransformation of toluene is accomplished not only through known metabolic routes such as toluene 3-monooxygenase (T3MO), toluene 2-monooxygenase (T2MO), toluene 4-monooxygenase (T4MO), toluene methyl monooxygenase (TOL), toluene dioxygenase (Tod), meta- and ortho-ring fission pathways. But additionally, confirmed existence of a unique metabolic pathway that involved conversion of toluene into intermediates such as cyclohexene, cyclohexane, cyclohexanone and cyclohexanol. LC-MS analysis indicated the presence of fatty acid amides, stigmine, emmotin A and 2, 2-dinitropropanol in supernatants of bacterial cultures. As the isolated bacteria transformed toluene into relatively less toxic molecules and thus can be preferably exploited for the eco-friendly remediation of toluene.

Ultrasensitive Detection of Cancer Cells Combining Enzymatic Signal Amplification with an Aerolysin Nanopore.

Sensitive detection of cancer cells at extremely low concentrations would greatly facilitate the screening and early diagnosis of cancer. Herein, we present a novel nanopore-based strategy for ultrasensitive detection of Ramos cells (human Burkitt's lymphoma cells), by combining the enzymatic signal amplification with an aerolysin nanopore sensor. In this assay, an aptamer for Ramos cells was prehybridized with a short complementary DNA. The presence of target cells causes the target-aptamer complex to unwind to free the complementary DNA, which would subsequently trigger the enzymatic cycling amplification. This process eventually generated a large number of output DNA, which could quantitatively produce characteristic current events when translocated through aerolysin. The proposed method exhibits excellent sensitivity, and as few as 5 Ramos cells could be detected. With good selectivity, the approach can allow for the determination of cancer cells in human serum, offering a powerful tool for biomedical research and clinical diagnosis.

Paclitaxel Biosynthesis: Adenylation and Thiolation Domains of an NRPS TycA PheAT Module Produce Various Arylisoserine CoA Thioesters.

Structure-activity relationship studies show that the phenylisoserinyl moiety of paclitaxel (Taxol) is largely necessary for the effective anticancer activity. Several paclitaxel analogues with a variant isoserinyl side chain have improved pharmaceutical properties versus those of the parent drug. To produce the isoserinyl CoAs as intermediates needed for enzyme catalysis on a semibiosynthetic pathway to paclitaxel analogues, we repurposed the adenylation and thiolation domains (Phe-AT) of a nonribosomal peptide synthetase (TycA) so that they would function as a CoA ligase. Twenty-eight isoserine analogue racemates were synthesized by an established procedure based on the Staudinger [2+2] cycloaddition reaction. Phe-AT converted 16 substituted phenylisoserines, one ß-(heteroaryl)isoserine, and one ß-(cyclohexyl)isoserine to their corresponding isoserinyl CoAs. We imagine that these CoA thioesters can likely serve as linchpin biosynthetic acyl donors transferred by a 13-O-acyltransferase to a paclitaxel precursor baccatin III to make drug analogues with better efficacy. It was also interesting to find that an active site mutant [Phe-AT (W227S)] turned over 2-pyridylisoserine and the sterically demanding p-methoxyphenylisoserine substrates to their CoA thioesters, while Phe-AT did not. This mutant is promising for further development to make 3-fluoro-2-pyridylisoserinyl CoA, a biosynthetic precursor of the oral pharmaceutical tesetaxel used for gastric cancers.

A thermostable pyrimidine nucleoside phosphorylase from Brevibacillus borstelensis LK01 for synthesizing halogenated nucleosides.

OBJECTIVE: To isolate a thermostable pyrimidine nucleoside phosphorylase (PyNP) from mesophilic bacteria by gene mining. RESULTS: BbPyNP from Brevibacillus borstelensis LK01 was isolated by gene mining. BbPyNP had a highest 60% identity with that of reported PyNPs. BbPyNP could catalyze the phosphorolysis of thymidine, 2'-deoxyuridine, uridine and 5-methyuridine. BbPyNP had good thermostability and retained 73% of its original activity after 2 h incubation at 50 °C. BbPyNP had the highest activity at an optimum alkaline pH of 8.5. BbPyNP was stable from pH 7 to 9.8. Under preliminary optimized conditions, the biosynthesis of various 5-halogenated pyrimidine nucleosides by BbPyNP reached the yield of 61-84%. CONCLUSION: An efficient approach was estimated in isolating thermostable PyNP from mesophilic bacteria.

Structural basis for DNA-mediated allosteric regulation facilitated by the AAA+ module of Lon protease.

Lon belongs to a unique group of AAA+ proteases that bind DNA. However, the DNA-mediated regulation of Lon remains elusive. Here, the crystal structure of the α subdomain of the Lon protease from Brevibacillus thermoruber (Bt-Lon) is presented, together with biochemical data, and the DNA-binding mode is delineated, showing that Arg518, Arg557 and Arg566 play a crucial role in DNA binding. Electrostatic interactions contributed by arginine residues in the AAA+ module are suggested to be important to DNA binding and allosteric regulation of enzymatic activities. Intriguingly, Arg557, which directly binds DNA in the α subdomain, has a dual role in the negative regulation of ATPase stimulation by DNA and in the domain-domain communication in allosteric regulation of Bt-Lon by substrate. In conclusion, structural and biochemical evidence is provided to show that electrostatic interaction in the AAA+ module is important for DNA binding by Lon and allosteric regulation of its enzymatic activities by DNA and substrate.

Production and purification of a hyperthermostable chitinase from Brevibacillus formosus BISR-1 isolated from the Great Indian Desert soils.

A strain of Brevibacillus formosus, capable of producing a high level of chitinase, was isolated and characterized for the first time from the Great Indian Desert soils. The production of extracellularly secreted chitinase was analyzed for its biocontrol potential and optimized by varying media pH, temperature, incubation period, substrate concentrations, carbon and nitrogen sources, etc. A twofold increase in chitinase production (798 IU/mL) was achieved in optimized media containing (g l(-1)) chitin 2.0, malt extract 1.5, glycerol 1.0, ammonium nitrate 0.3%, T-20 (0.1%) and media pH 7.0 at 37 °C. The produced enzyme was purified using a three-step purification procedure involving ultra-filtration, ammonium sulphate precipitation and adsorption chromatography. The estimated molecular weight of the purified enzyme was 37.6 kDa. The enzyme was found thermostable at higher temperatures and showed a t ½ of more than 5 h at 100 °C. Our results show that the chitinase produced by B. formosus BISR-1 is thermostable at higher temperatures.

Structure and engineering of Brevibacillus laterosporus Cas9.

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an unexpected architecture containing a triple-helix, which is specifically recognized by BlCas9, and that BlCas9 recognizes a unique N4CNDN protospacer adjacent motif through base-specific interactions on both the target and non-target DNA strands. Based on the structure, we rationally engineered a BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target scope in human cells. This approach may further improve the performance of the enhanced BlCas9 variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can be packaged into a single AAV vector for in vivo gene therapy.

Efficient soluble expression of secreted matrix metalloproteinase 26 in Brevibacillus choshinensis.

Matrix metalloproteinase 26 (MMP-26) is a novel member of the matrix metalloproteinase family with minimal domain constitution and unknown physiological function. The three-dimensional (3D) structure of the enzyme also remains to be deciphered. Previous studies show that MMP-26 may be expressed in Escherichia coli (E. coli) as inclusion bodies and re-natured with catalytic activity. However, the low re-naturation rate of this method limits its usage in structural studies. In this paper, we tried to clone, express and purify the pro form and catalytic form of MMP-26 (ProMMP-26 and CatMMP-26) in several widely used expression vectors and express the recombinant MMP-26 proteins in E. coli cells. These constructs resulted in insoluble expressions or soluble expressions of MMP-26 with little catalytic activity. We then used Brevibacillus choshinensis (B. choshinensis) as the host system for the soluble and active expression of MMP-26. The enzyme was secreted in soluble form in the supernatant of cell culture medium and purified via a two-step purification process that included Ni(2+) affinity chromatography followed by gel filtration. The yields of purified ProMMP-26 and CatMMP-26 were 12 and 18mg/L, respectively, with high purity and homogeneity. Both ProMMP-26 and CatMMP-26 showed gelatin zymography activity and the purified CatMMP-26 had high enzymatic activity against DQ-gelatin substrate. The large-scale soluble and active protein production for future structural studies of MMP-26 is thus feasible using the B. choshinensis host system.

Crystallization and preliminary X-ray diffraction analysis of the α subdomain of Lon protease from Brevibacillus thermoruber.

DNA-binding ability has previously been reported as a novel function for the thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon), and the α subdomain (amino acids 491-605) of Bt-Lon has been identified as being responsible for DNA binding. However, the physiological role and DNA-recognition mode of Bt-Lon still remain unclear. In this study, the crystallization and preliminary crystallographic analysis of the Bt-Lon α subdomain are presented. Native diffraction data to 2.88 Å resolution were obtained from a vitrified crystal at 100 K on the BL13C1 beamline at the NSRRC (National Synchrotron Radiation Research Center), Taiwan. The crystals belonged to space group P23, with unit-cell parameters a = b = c = 94.28 Å. Solvent-content calculations and molecular-replacement results suggest that there are two molecules of Bt-Lon α subdomain per asymmetric unit.

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential.

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain's antifungal activity and insecticidal activity.

Trimeric l-N-carbamoylase from newly isolated Brevibacillus reuszeri HSN1: a potential biocatalyst for production of l-α-amino acids.

l-N-carbamoylase was isolated from Brevibacillus reuszeri HSN1 and purified to homogeneity in three steps, which is a reasonably short protocol for native l-N-carbamoylase. The enzyme purification protocol resulted in ≈60-fold purification of l-N-carbamoylase with specific activity of 145 µmol/Min/mg. The subunit and native molecular mass were found to be 44.3 and 132 kDa, respectively. Temperature and pH optima were determined as 50°C and 8.5, respectively. The enzyme had retained ≈86% activity at 50°C when incubated for 60 Min and the half-life was determined as 180 Min at 50°C. N-carbamoyl-l-methionine (l-N-CMet) was found to be a preferred substrate with Km and Vmax values of ≈13.5 mM and ≈103 µmol/Min/mg, respectively. The broad substrate specificity with derivatives of N-carbamoyl amino acids is advantageous to be a better biocatalyst for production of corresponding l-α-amino acids. The enzyme activity was enhanced by 73% in the presence of 0.8 mM Mn(2+) ion during the biotransformation. In the batch experiment, ≈97% conversion of 5.0% l-N-CMet into enantiomerically pure l-methionine was achieved in 10 H when carried out at pH 8.0, 45°C, and 15% wet (w/v) cell loading, under controlled conditions. The overall merits of this enzyme show promise as a potential biocatalyst for l-α-amino acid production.

Characterization of endo-1,3-1,4-ß-glucanases in GH family 12 from Magnaporthe oryzae.

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze ß-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3-1,4-ß-glucans, such as barley ß-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3-1,4-ß-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3-1,4-ß-glucan during infection and spore formation.

Production of an in vitro-derived deletion mutation of Brevibacillus laterosporus by constructing a homology-driven integration vector.

In this study, a homology-driven integration vector and electroporation system was developed to delete a protease gene in the pathogenic bacterium Brevibacillus laterosporus strain G4. Furthermore, an in vitro protease-deficient mutation was generated by introducing the integration vector with a 445-bp protease BLG4 fragment into B. laterosporus chromosomal target via homologous recombination. The BLG4-deficient mutant showed a significant drop in protease activity as compared to the wild-type G4 strain, but had a slight effect on bacterial growth and sporulation. The results revealed that the developed method can become an important tool for studying the molecular pathogenesis mechanisms of B. laterosporus.

Sustainable Denim Bleaching by a Novel Thermostable Bacterial Laccase.

Laccases have emerged as environment-friendly multifaceted biocatalysts for diverse biotechnological applications. Here, we isolated a high molecular weight (88 kDa) extremophilic laccase (LacT) from Brevibacillus agri, with the aim to exploit its extreme characters in denim bleaching. LacT has been characterized as a thermostable, acidophilic enzyme with high salt, organic solvent, and divalent metal tolerance properties. Denim bleaching efficiency of LacT was optimum at pH 4.0 and appeared to be surpassing over other reported laccases. LacT also exhibited remarkable efficacy in the decolorization of water-soluble health hazardous azo-dyes, and thus transpired to be a promising bio-bleaching and dye decolorizing agent.

A novel thermal Cas12b from a hot spring bacterium with high target mismatch tolerance and robust DNA cleavage efficiency.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas), such as Cas9 and Cpf1, are RNA-guided endonucleases that target and degrade nucleic acids, providing powerful genomic editing and molecular diagnostic tools. Cas12b enzymes are distinct effectors; however, their features and catalytic boundaries require further characterization. We identified BrCas12b from the thermophile bacterium Brevibacillus sp. SYSU G02855 as a novel ortholog of cas12b. Biochemical analyses revealed that BrCas12b is a dual-RNA-guided endonuclease with higher optimum reaction temperature than that of other reported members of Cas12b. The seed sequence of BrCas12b is only 4 nt in length, indicating that it has greater target mismatch tolerance than that of previously reported Cas effectors; however, it contains a compensatory effect at the position of the cleavage site. Using fluorescence-based detection method to evaluate target cleavage efficiency, we showed that BrCas12b has robust enzymatic cleavage activity (Kcat/Km (s-1 M-1) = 8.80 × 1011), which is significantly higher than that of AacCas12b (Kcat/Km (s-1 M-1) = 7.56 × 108) from Alicyclobacillus acidoterrestris. The results increase our understanding of the catalytic mechanism of Cas12b family members and suggest that BrCas12b might be useful in the application of genomic editing and molecular diagnosis.

Utilization of methyltrioctylammonium chloride as new ionic liquid in pretreatment of sugarcane bagasse for production of cellulase by novel thermophilic bacteria.

Fermentation of carbohydrates present in lignocellulosic (LC) biomass is facilitated by lignin removal, which is usually achieved by adopting various pretreatment methods to provide the enzymes proper access to their respective substrates. Pretreatment using ionic liquid (IL) is relatively recent advancement and considered as mild and green process. ILs can dissolve extensive quantities of biomass and depolymerize the cellulose. In this context, an abundantly available LC biomass, sugarcane bagasse (SB), was pretreated using alkali or with an IL, methyltrioctylammonium chloride, and was used for cellulase production from thermophilic bacteria. In all, 26 indigenously isolated thermophilic bacterial strains were quantitatively screened for cellulase production. 16S rDNA sequences of the promising isolates UE10 and UE27 revealed relatedness with Brevibacillus borstelensis, while the strain UE1 belonged to Aneurinibacillus thermoaerophilus. Cellulase production was compared by utilizing alkali pretreated and IL pretreated SB and the later was found more appropriate. UE1, UE10 and UE27 yielded 22.2, 22.18 and 33.3 IU mL-1 of endoglucanase, respectively, by fermenting IL pretreated SB. The changes in SB structure after pretreatment were evaluated by scanning electron microscopy. This study demonstrated the potential of novel thermophilic bacterial strains to utilize IL pretreated SB for production of industrially important enzyme, cellulase.

Recombinant expression and molecular engineering of the keratinase from Brevibacillus parabrevis for dehairing performance.

Keratinase is capable of distinctive degradation of keratin, which provides an eco-friendly approach for keratin waste management towards sustainable development. In this study, the recombinant keratinase (KERBP) from Brevibacillus parabrevis was successfully expressed in Escherichia coli. The purified KERBP had the specific activity of 6005.3 U/mg. It showed remarkable tolerance to various surfactants and also no collagenolytic activity. However, the moderate thermal stability limited its further application. Thus, protein engineering was further adopted to improve its stability. The variants of T218S, S236C and N181D were constructed by site-directed mutagenesis and combinatorial mutagenesis. Compared with the wild type, the t1/2 at 60 °C for the variants T218S, S236C and N181D were 3.05-, 1.18- and 1-fold increase, respectively. Moreover, the double variants N181D-T218S and N181D-S236C significantly improved thermostability with 5.1 and 2.9 °C increase of T50, and prolonging t1/2 at 60 °C with 4.09 and 1.54-fold, respectively. And the catalytic efficiency of the T218S and N181D-T218S variants was also significantly improved. Furthermore, the keratinase displayed favorable ability to dehair wool from skin within 7 h, which showed potential in leather dehairing. Our work contributes to a further insight into the thermostability of keratinase and offers a promising alternative for industrial leather application.

Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility.

Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.