Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps.

Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA-bound proteins, and basic properties like the elementary step size remain controversial. Using single-molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single-stranded loop. Static disorder limited previous ensemble studies of a PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations, suggesting a mode of action for eliminating potentially deleterious recombination intermediates.

Polyhydroxyalkanoate biosynthesis and optimisation of thermophilic Geobacillus stearothermophilus strain K4E3_SPR_NPP.

Polyhydroxyalkanoates (PHA) can be used to combat the challenges associated with plastic because it is biodegradable and can be produced from renewable resources. Extremophiles are considered to be potential PHA producers. An initial screening for the PHA synthesizing ability of a thermophilic bacteria Geobacillus stearothermophilus strain K4E3_SPR_NPP was carried out using Sudan black B staining. Nile red viable colony staining was used to further verify that the isolates produced PHA. Crotonic acid assays were used to determine the concentrations of PHA. The bacteria showed 31% PHA accumulation per dry cell weight (PHA/DCW) when glucose was used as a carbon source for growth. The molecule was identified to be medium chain length PHA, A copolymer of PHA containing poly(3-hydroxybutyrate)-poly(3-hydroxyvalerate)-poly(3-hydroxyhexanoate) (PHB-PHV-PHHX) using 1H-NMR. Six carbon sources and four nitrogen sources were screened for the synthesis of maximum PHA content, of which lactose and ammonium nitrate showed 45% and 53% PHA/DCW respectively. The important factors in the experiment are identified using the Plackett-Burman design, and optimization is performed using the response surface method. Response surface methodology was used to optimize the three important factors, and the maximum biomass and PHA productions were discovered. Optimal concentrations yielded a maximum of 0.48 g/l biomass and 0.32 g/l PHA, measuring 66.66% PHA accumulation. Dairy industry effluent was employed for the synthesis of PHA, yielding 0.73 g/l biomass and 0.33 g/l PHA, measuring 45% PHA accumulation. These findings add credibility to the possibility of adopting thermophilic isolates for PHA production using low-cost substrates.

Effect of solid-state fermentation by three different Bacillus species on composition and protein structure of soybean meal.

BACKGROUND: Fermentation efficiency of thermophiles of Bacillus licheniformis YYC4 and Geobacillus stearothermophilus A75, and mesophilic Bacillus subtilis 10 160 on soybean meal (SBM), was evaluated by examining the nutritional and protein structural changes. RESULTS: SBM fermentation by B. licheniformis YYC4, B. subtilis 10 160 and G. stearothemophilus A75 increased significantly the crude and soluble protein from 442.4 to 524.8, 516.1 and 499.9 g kg-1 , and from 53.9 to 203.3, 291.3 and 74.6 g kg-1 , and decreased trypsin inhibitor from 8.19 to 3.19, 2.14 and 5.10 mg g-1 , respectively. Bacillus licheniformis YYC4 and B. subtilis 10 160 significantly increased phenol and pyrazine content. Furthermore, B. licheniformis YYC4 fermentation could produce abundant alcohols, ketones, esters and acids. Surface hydrophobicity, sulfhydryl groups and disulfide bond contents of SBM protein were increased significantly from 98.27 to 166.13, 173.27 and 150.71, from 3.26 to 4.88, 5.03 and 4.21 µmol g-1 , and from 20.77 to 27.95, 29.53 and 25.5 µmol g-1 after their fermentation. Fermentation induced red shifts of the maximum absorption wavelength (λmax ) of fluorescence spectra from 353 to 362, 376 and 361 nm, while significantly reducing the fluorescence intensity of protein, especially when B. subtilis 10 160 was used. Moreover, fermentation markedly changed the secondary structure composition of SBM protein. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and atomic force microscopy showed that macromolecule protein was degraded into small-sized protein or peptide during fermentation of SBM. CONCLUSION: Bacillus licheniformis YYC4 fermentation (without sterilization) improved nutrition and protein structure of SBM as B. subtilis 10 160, suggesting its potential application in the SBM fermentation industry. © 2021 Society of Chemical Industry.

Ribosome-Amplified Metabolism, RAMBO, Measured by NMR Spectroscopy.

NMR spectroscopy was used to investigate the phenomenon of ribosome-amplified metabolism or RAMBO between pyruvate kinase and ribosomes. Because the concentration of ribosomes increases as the cell grows, ribosome binding interactions may regulate metabolic fluxes by altering the distribution of bound and free enzymes. Pyruvate kinase (PK) catalyzes the last step of glycolysis and represents a major drug target for controlling bacterial infections. The binding of metabolic enzymes to ribosomes creates protein quinary structures with altered catalytic activities. NMR spectroscopy and chemical cross-linking combined with high-resolution mass spectrometry were used to establish that PK binds to ribosome at three independent sites, the L1 stalk, the A site, and the mRNA entry pore. The bioanalytical methodology described characterizes the altered kinetics and confirms the specificity of pyruvate kinase-ribosome interaction, affording an opportunity to investigate the ribosome dependence of metabolic reactions under solution conditions that closely mimic the cytosol. Expanding on the concept of ribosomal heterogeneity, which describes variations in ribosomal constituents that contribute to the specificity of cellular processes, this work firmly establishes the reciprocal process by which ribosome-dependent quinary interactions affect metabolic activity.

Cross-utilization of ß-galactosides and cellobiose in Geobacillus stearothermophilus.

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-ß-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-ß-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

Comparison of conventional and extractive fermentation using aqueous two-phase system to extract fibrinolytic proteases produced by Bacillus stearothermophilus DPUA 1729.

Fibrinolytic enzymes have been considered promising for treatment and protection of healthy circulation due its ability to dissolve the fibrin in blood clots. Extractive fermentation is a not explored and efficient downstream process which segregates the desired product simultaneously in a fermentation process fast and economically. Extraction of fibrinolytic enzymes by Bacillus stearothermophilus DPUA 1729 employing conventional aqueous two-phase systems (ATPS) and extractive fermentation with ATPS was evaluated. The results of both systems were compared using a factorial design with PEG molar mass, PEG and salt concentrations as independent variables and extraction parameters as a response. In all conditions evaluated it was observed a similar partitioning of fibrinolytic enzymes through the phases, both in conventional ATPS and extractive fermentation. Salt concentration and interaction among PEG and salt concentration influenced in the partition coefficient. The fibrinolytic activity was determined by hydrolysis of fibrin in plate using the extract of one condition from extractive fermentation. The zone degradation presented a diameter of 7.03 ± 0.94 mm. In conclusion, there was no significant difference among the results obtained using conventional ATPS and extractive fermentation, however, the second one presents more advantages and can integrate production and extraction in one single step, reducing the costs.

Effect of Different Proportions of Three Microbial Agents on Ammonia Mitigation during the Composting of Layer Manure.

Odor emissions represent one of the important issues of aerobic composting. The addition of microbial agents to compost is an important method for solving this problem, but this process is often unstable when a single microbial agent is added to the compost. Therefore, in this study, five treatments comprising different proportions of Bacillus stearothermophilus, Candida utilis, and Bacillus subtilis were tested to determine the best combination of the three microbial agents for ammonia reduction, as follows: control group (CK), 2:1:1 (A), 1:1:2 (B), 1:2:1 (C), and 1:1:1 (D). Compared with the CK group, the A, B, C, and D groups reduced ammonia emissions by 17.02, 9.68, 53.11, and 46.23%, respectively. The total ammonia emissions were significantly lower in C and D than in CK (p < 0.05). These two treatment groups had significantly increased nitrate nitrogen concentrations and decreased pH values and ammonium nitrogen concentrations (p < 0.05). Throughout the composting process, the total bacterial number was significantly higher in C and D than in CK (p < 0.05). Therefore, it is likely that B. stearothermophilus, C. utilis, and B. subtilis compounded from 1:2:1 (C) to 1:1:1 (D) reduced the ammonia emissions due to (1) a reduction in the pH and (2) the promotion of the growth of ammonia-oxidizing bacteria and the conversion of ammonium nitrogen to nitrate nitrogen. This study provides a theoretical basis and technical support for the odor problem of layer manure compost and promotes the development of composting technology.

Feasibility study on direct fermentation of soybean meal by Bacillus stearothermophilus under non-sterile conditions.

BACKGROUND: To evaluate the feasibility of high-temperature solid-state fermentation (SSF) using soybean meal (SBM) during the non-sterile process, Bacillus stearothermophilus was employed to assess the nutritional quality and bioactivity of SBM after fermentation. RESULTS: The fermented SBM (FSBM) without autoclaving showed significant improvements in nutritional quality and bioactivity. The contents of peptides and crude and soluble proteins increased by 131.21%, 5.3% and 15.52%, respectively. Meanwhile, DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging ability, reducing ability and hydroxyl free radical-scavenging activity rose by 57.07%, 238.92% and 368.26%, respectively. The inhibitory activity of angiotensin I-converting enzyme increased from 1.43 ± 0.83% to 26.89 ± 1.03%, while the trypsin inhibitor activity decreased by 74.05%. The contents of neutral and alkaline proteases and the growth of microorganisms in FSBM without autoclaving were higher and better than in steam-treated FSBM. After steam treatment, the water-holding capacity of SBM decreased, and a high crosslink density was observed on the surface of SBM particles. CONCLUSIONS: It is feasible to ferment SBM by high-temperature SSF using B. stearothermophilus under non-sterile conditions. Adverse effects of SSF using sterile SBM might be owing to the low water-holding capacity caused by autoclaving. © 2018 Society of Chemical Industry.

One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP)2 of Geobacillus stearothermophilus.

ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN)2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243-5248, 2015, https://doi.org/10.1073/pnas.1415037112). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP)2 transporter of Geobacillus stearothermophilus, by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P]2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer.IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus, encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

Structural Insight into the Discrimination between 8-Oxoguanine Glycosidic Conformers by DNA Repair Enzymes: A Molecular Dynamics Study of Human Oxoguanine Glycosylase 1 and Formamidopyrimidine-DNA Glycosylase.

hOgg1 and FPG are the primary DNA repair enzymes responsible for removing the major guanine (G) oxidative product, namely, 7,8-dihydro-8-oxoguanine (OG), in humans and bacteria, respectively. While natural G adopts the anti conformation and forms a Watson-Crick pair with cytosine (C), OG can also adopt the syn conformation and form a Hoogsteen pair with adenine (A). hOgg1 removes OG paired with C but is inactive toward the OG:A pair. In contrast, FPG removes OG from OG:C pairs and also exhibits appreciable (although diminished) activity toward OG:A pairs. As a first step toward understanding this difference in activity, we have employed molecular dynamics simulations to examine how the anti and syn conformers of OG are accommodated in the hOgg1 and FPG active sites. When anti-OG is bound, hOgg1 active site residues are properly aligned to initiate catalytic base departure, while geometrical parameters required for the catalytic reaction are not conserved for syn-OG. On the other hand, the FPG catalytic residues are suitably aligned for both OG conformers, with anti-OG being more favorably bound. Thus, our data suggests that the differential ability of hOgg1 and FPG to accommodate the anti- and syn-OG glycosidic conformations is an important factor that contributes to the relative experimental excision rates. Nevertheless, the positions of the nucleophiles with respect to the lesion in the active sites suggest that the reactant complex is poised to initiate catalysis through a similar mechanism for both repair enzymes and supports a recently proposed mechanism in which sugar-ring opening precedes nucleoside deglycosylation.

Structural Basis for the Lesion-scanning Mechanism of the MutY DNA Glycosylase.

The highly mutagenic A:8-oxoguanine (oxoG) base pair is generated mainly by misreplication of the C:oxoG base pair, the oxidation product of the C:G base pair. The A:oxoG base pair is particularly insidious because neither base in it carries faithful information to direct the repair of the other. The bacterial MutY (MUTYH in humans) adenine DNA glycosylase is able to initiate the repair of A:oxoG by selectively cleaving the A base from the A:oxoG base pair. The difference between faithful repair and wreaking mutagenic havoc on the genome lies in the accurate discrimination between two structurally similar base pairs: A:oxoG and A:T. Here we present two crystal structures of the MutY N-terminal domain in complex with either undamaged DNA or DNA containing an intrahelical lesion. These structures have captured for the first time a DNA glycosylase scanning the genome for a damaged base in the very first stage of lesion recognition and the base extrusion pathway. The mode of interaction observed here has suggested a common lesion-scanning mechanism across the entire helix-hairpin-helix superfamily to which MutY belongs. In addition, small angle X-ray scattering studies together with accompanying biochemical assays have suggested a possible role played by the C-terminal oxoG-recognition domain of MutY in lesion scanning.

Sulfur K-Edge XAS Studies of the Effect of DNA Binding on the [Fe4S4] Site in EndoIII and MutY.

S K-edge X-ray absorption spectroscopy (XAS) was used to study the [Fe4S4] clusters in the DNA repair glycosylases EndoIII and MutY to evaluate the effects of DNA binding and solvation on Fe-S bond covalencies (i.e., the amount of S 3p character mixed into the Fe 3d valence orbitals). Increased covalencies in both iron-thiolate and iron-sulfide bonds would stabilize the oxidized state of the [Fe4S4] clusters. The results are compared to those on previously studied [Fe4S4] model complexes, ferredoxin (Fd), and to new data on high-potential iron-sulfur protein (HiPIP). A limited decrease in covalency is observed upon removal of solvent water from EndoIII and MutY, opposite to the significant increase observed for Fd, where the [Fe4S4] cluster is solvent exposed. Importantly, in EndoIII and MutY, a large increase in covalency is observed upon DNA binding, which is due to the effect of its negative charge on the iron-sulfur bonds. In EndoIII, this change in covalency can be quantified and makes a significant contribution to the observed decrease in reduction potential found experimentally in DNA repair proteins, enabling their HiPIP-like redox behavior.

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly.

Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5' domain of the 16S rRNA. In vitro, transformation of initial S4-rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4-rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein-RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4-RNA interactions guide rRNA folding and impact late steps of 30S assembly.

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

Unexpected non-Hoogsteen-based mutagenicity mechanism of FaPy-DNA lesions.

8-Oxopurines (8-oxodG and 8-oxodA) and formamidopyrimidines (FaPydG and FaPydA) are major oxidative DNA lesions involved in cancer development and aging. Their mutagenicity is believed to result from a conformational shift of the N9-C1' glycosidic bonds from anti to syn, which allows the lesions to form noncanonical Hoogsteen-type base pairs with incoming triphosphates during DNA replication. Here we present biochemical data and what are to our knowledge the first crystal structures of carbocyclic FaPydA and FaPydG containing DNA in complex with a high-fidelity polymerase. Crystallographic snapshots show that the cFaPy lesions keep the anti geometry of the glycosidic bond during error-free and error-prone replication. The observed dG·dC→dT·dA transversion mutations are the result of base shifting and tautomerization.

Residues of a proposed gate region in type I ATP-binding cassette import systems are crucial for function as revealed by mutational analysis.

The type I ATP-binding cassette (ABC) importer for positively charged amino acids of the thermophilic bacterium Geobacillus stearothermophilus consists of the extracellular solute binding protein, ArtJ, and a homodimer each of the transmembrane subunit, ArtM, and the nucleotide-binding and -hydrolyzing subunit, ArtP. We have investigated the functional consequences of mutations affecting conserved residues from two peptide regions in ArtM, recently proposed to form a 'gate' by which access of a substrate to the translocation path is controlled (Hollenstein et al., 2007 [14]). Transporter variants were reconstituted into proteoliposomes and assayed for ArtJ/arginine-stimulated ATPase activity. Replacement of residues from region 1 (Arg-63, Pro-66) caused no or only moderate reduction in ATPase activity. In contrast, mutating residues from gate region 2 (Lys-159, Leu-163) resulted in a substantial increase in ATPase activity which, however, as demonstrated for variants ArtM(K159I) and ArtM(K159E), is not coupled to transport. Replacing homologous residues in the closely related histidine transporter of Salmonella enterica serovar Typhimurium (HisJ-QMP2) caused different phenotypes. Mutation to isoleucine of HisQ(K163) or HisM(H172), both homologous to ArtM(K159), abolished ATPase activity. The mutations most likely caused a structural change as revealed by limited proteolysis. In contrast, substantial, albeit reduced, enzymatic activity was observed with variants of HisQ(L167→G) or HisM(L176→G), both homologous to ArtM(L163). Our study provides the first experimental evidence in favor of a crucial role of residues from the proposed gate region in type I ABC importer function.

Structure-specificity relationships in Abp, a GH27 ß-L-arabinopyranosidase from Geobacillus stearothermophilus T6.

L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system in Geobacillus stearothermophilus T6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular ß-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove ß-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Šresolution) and its catalytic mutant Abp-D197A with (at 2.20 Šresolution) and without (at 2.30 Šresolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-ß domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Šfrom each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer made up of two `open-pincers' dimers, which clamp around each other to form a central cavity. The four active sites of the Abp tetramer are situated on the inner surface of this cavity, all opening into the central space of the cavity. The biological relevance of this tetrameric structure is supported by independent results obtained from size-exclusion chromatography (SEC), dynamic light-scattering (DLS) and small-angle X-ray scattering (SAXS) experiments. These data and their comparison to the structural data of related GH27 enzymes are used for a more general discussion concerning structure-selectivity aspects in this glycoside hydrolase (GH) family.

Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering.

Metabolic engineers develop inexpensive enantioselective syntheses of high-value compounds, but their designs are sometimes confounded by the misfolding of heterologously expressed proteins. Geobacillus stearothermophilus NUB3621 is a readily transformable facultative thermophile. It could be used to express and properly fold proteins derived from its many mesophilic or thermophilic Bacillaceae relatives or to direct the evolution of thermophilic variants of mesophilic proteins. Moreover, its capacity for high-temperature growth should accelerate chemical transformation rates in accordance with the Arrhenius equation and reduce the risks of microbial contamination. Its tendency to sporulate in response to nutrient depletion lowers the costs of storage and transportation. Here, we present a draft genome sequence of G. stearothermophilus NUB3621 and describe inducible and constitutive expression plasmids that function in this organism. These tools will help us and others to exploit the natural advantages of this system for metabolic engineering applications.

Crystal structure and solution scattering of Geobacillus stearothermophilus S9 peptidase reveal structural adaptations for carboxypeptidase activity.

Acylaminoacyl peptidases (AAPs) play a pivotal role in various pathological conditions and are recognized as potential therapeutic targets. AAPs exhibit a wide range of activities, such as acylated amino acid-dependent aminopeptidase, endopeptidase, and less studied carboxypeptidase activity. We have determined the crystal structure of an AAP from Geobacillus stearothermophilus (S9gs) at 2.0 Å resolution. Despite being annotated as an aminopeptidase in the NCBI database, our enzymatic characterization proved S9gs to be a carboxypeptidase. Solution-scattering studies showed that S9gs exists as a tetramer in solution, and crystal structure analysis revealed adaptations responsible for the carboxypeptidase activity of S9gs. The findings present a hypothesis for substrate selection, substrate entry, and product exit from the active site, enriching our understanding of this rare carboxypeptidase.