Kinetic mechanism of phenylacetone monooxygenase from Thermobifida fusca

Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 73 s (-1)). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s (-1)) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP (+) release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.     

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP⁺, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.     

Chitin binding by Thermobifida fusca cellulase catalytic domains

Cellulose is a linear homopolymer of beta 1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase catalytic domains (CDs) to chitin has not been reported. In this article, binding of the CDs of Thermobifida fusca Cel6A, Cel6B, Cel48A, Cel5A, and Cel9A to alpha-chitin was investigated. The CDs of endocellulases, Cel6A and Cel5A did not bind to alpha-chitin; one exocellulase, Cel48A CD bound alpha-chitin moderately well; and the exocellulase Cel6B CD and the processive endocellulase Cel9A CD bound extremely tightly to alpha-chitin. Only mutations of Cel6B W329C, W332A and G234S and Cel9A Y206F, Y206S and D261A/R378K caused weaker binding to alpha-chitin than wild-type, and all these mutations were of residues near the catalytic center. One mutant enzyme, Cel9A D261A/R378K had weak chitinase activity, but no soluble products were detected. Chitotriose and chitotetraose were docked successfully to the catalytic cleft of Cel9A. In general, the positioning of the sugar residues in the model structures matched the cellooligosaccharides in the X-ray structure. Our results show that the binding of chitin by a cellulase can provide additional information about its binding to cellulose.     

Crystal structure of Thermobifida fusca Cse1 reveals target DNA binding site

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐CRISPR‐associated (Cas) defense system is the only adaptive and inheritable immunity found in prokaryotes. The immunity is achieved through a multistep process of adaptation, expression, and interference. In the Type I‐E system, interference is mediated by the CRISPR‐associated complex for antiviral defense (Cascade), which recognizes invading double‐stranded DNA (dsDNA) through the protospacer adjacent motif (PAM) by one of the Cascade components, Cse1. Here, we report the crystal structure of Thermobifida fusca Cse1 at 3.3 Å resolution. T. fusca Cse1 reveals the chair‐like two‐domain architecture with a well‐defined flexible loop, L1, located at the larger N‐terminal domain, which was not observed in previous structures of the single Cse1 protein. Structure‐based mutagenesis analysis demonstrates that the well‐defined flexible loop and a partially conserved structural motif ([FW]‐X‐[TH]) are involved in PAM binding and recognition, respectively. Moreover, structural docking of T. fusca Cse1 into Escherichia coli Cascade cryoelectron microscopy maps, coupled with structural comparison, reveals a conserved positive patch that is contiguous with Cse2 in the Cascade complex and adjacent to the Cas3 binding site, suggesting its role in R‐loop formation/stabilization and the recruitment of Cas3 for target cleavage. Consistent with the structural observation, the introduction of alanine mutations at this positive patch abolished DNA binding activity by Cse1. Taken together, these results suggest that Cse1 is a critical Cascade component involved in Cascade assembly, dsDNA target recognition, R‐loop formation, and Cas3 recruitment for target cleavage.     

Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(-1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(-2) to Glc(-4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(-2) to Glc(-4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.     

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

Biochemical characterization of the cutinases from Thermobifida fusca

Thermobifida fusca produces two cutinases which share 93% identity in amino acid sequence. In the present study, we investigated the detailed biochemical properties of T. fusca cutinases for the first time. For a better comparison between bacterial and fungal cutinases, recombinant Fusarium solani pisi cutinase was subjected to the similar analysis. The results showed that both bacterial and fungal cutinases are monomeric proteins in solution. The bacterial cutinases exhibited a broad substrate specificity against plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoenzymes of T. fusca and the F. solani pisi cutinase are similar in substrate kinetics, the lack of interfacial activation, and metal ion requirements. However, the T. fusca cutinases showed higher stability in the presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good tolerance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca cutinases, they may have promising applications in related industries.     

Structure and functional analysis of the siderophore periplasmic binding protein from the fuscachelin gene cluster of Thermobifida fusca

Iron acquisition is a complex, multicomponent process critical for most organisms' survival and virulence. Small iron chelating molecules, siderophores, mediate transport as key components of common pathways for iron assimilation in many microorganisms. The chemistry and biology of the extraordinary tight and specific metal binding siderophores is of general interest in terms of host/guest chemistry and is a potential target toward the development of therapeutic treatments for microbial virulence. The siderophore pathway of the moderate thermophile, Thermobifida fusca, is an excellent model system to study the process in Gram‐positive bacteria. Here we describe the structure and characterization of the siderophore periplasmic binding protein, FscJ from the fuscachelin gene cluster of T. fusca. The structure shows a di‐domain arrangement connected with a long α‐helix hinge. Several X‐ray structures detail ligand‐free conformational changes at different pH values, illustrating complex interdomain flexibility of the siderophore receptors. We demonstrated that FscJ has a unique recognition mechanism and details the binding interaction with ferric‐fuscachelin A through ITC and docking analysis. The presented work provides a structural basis for the complex molecular mechanisms of siderophore recognition and transportation. Proteins 2016; 84:118–128. © 2015 Wiley Periodicals, Inc.     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca

The gene coding for a hemoglobin-like protein (Tf-trHb) has been identified in the thermophilic actinobacterium Thermobifida fusca and cloned in Escherichia coli for overexpression. The crystal structure of the ferric, acetate-bound derivative, was obtained at 2.48 A resolution. The three-dimensional structure of Tf-trHb is similar to structures reported for the truncated hemoglobins from Mycobacterium tuberculosis and Bacillus subtilis in its central domain. The complete lack of diffraction patterns relative to the N- and C-terminal segments indicates that these are unstructured polypeptides chains, consistent with their facile cleavage in solution. The absence of internal cavities and the presence of two water molecules between the bound acetate ion and the protein surface suggest that the mode of ligand entry is similar to that of typical hemoglobins. The protein is characterized by higher thermostability than the similar mesophilic truncated hemoglobin from B. subtilis, as demonstrated by far-UV CD melting experiments on the cyano-met derivatives. The ligand-binding properties of Tf-trHb, analyzed in stopped flow experiments, demonstrate that Tf-trHb is capable of efficient O2 binding and release between 55 and 60 degrees C, the optimal growth temperature for Thermobifida fusca.     

Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes

Thermobifida fusca Cel9A-90, an unusual family 9 enzyme, is a processive endoglucanase containing a catalytic domain closely linked to a family 3c cellulose binding domain (Cel9A-68) followed by a fibronectin III-like domain and a family 2 cellulose binding domain. To study its catalytic mechanism, 12 mutant genes with changes in five conserved residues of Cel9A-68 were constructed, cloned, and expressed in Escherichia coli. The purified mutant enzymes were assayed for their activities on (carboxymethyl)cellulose, phosphoric acid-swollen cellulose, bacterial microcrystalline cellulose, and 2,4-dinitrophenyl beta-D-cellobioside. They were also tested for ligand binding, enzyme processivity, and thermostability. The results clearly show that E424 functions as the catalytic acid, D55 and D58 are both required for catalytic base activity, and Y206 plays an important role in binding, catalysis, and processivity, while Y318 plays an important role in binding of crystalline cellulose substrates and is required for processivity. Several amino acids located in a loop at the end of the catalytic cleft (T245-L251) were deleted from Cel9A-68, and this enzyme showed slightly improved filter paper activity and binding to BMCC but otherwise behaved like the wild-type enzyme. The FnIII-like domain was deleted from Cel9A-90, reducing BMCC activity to 43% of the wild type.     

Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase cel6A.

The availability of a high-resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure-based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active-site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand-binding affinity. Eleven mutations were generated by site-directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM-cellulose), with K259H (in glucosyl subsite -2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM-cellulose activity. Only the K259H enzyme retained more activity on acid-swollen cellulose than on filter paper, suggesting that this mutation affected the rate-limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6-fold and 5.0-fold, respectively) than Cel6A on 2,4-dinitrophenyl-beta-D-cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity.     

产几丁质酶褐色喜热裂孢菌的分离鉴定及产酶特性初步研究

采用选择性培养基从土壤中分离到1株产几丁质酶的微生物菌株YX,经形态和分子鉴定为褐色喜热裂孢菌(Thermobifida fusca)。进一步在摇瓶中比较了T.fusca YX在纤维二糖、几丁质、或羧甲基纤维素钠为碳源的培养基中的产酶特性,YX菌株在5 L发酵罐中以几丁质为碳源的培养基发酵到22 h左右时发酵液几丁质酶活即可达到1.7 U/m L。本文首次报道褐色喜热裂孢菌能够产生几丁质酶,具有潜在的应用价值。     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

Mapping the substrate binding site of the prostaglandin transporter PGT by cysteine scanning mutagenesis.

We have identified a cDNA, PGT, that encodes a widely expressed transporter for prostaglandin (PG) E(2), PGF(2alpha), PGD(2), 8-iso-PGF(2alpha), and thromboxane B(2). To begin to understand the molecular mechanisms of transporter function, we have initiated a structure-function analysis of PGT to identify its substrate-binding region. We have found that by introducing the small, water-soluble, thiol-reactive anion Na(2-sulfonatoethyl)methanethiosulfonate (MTSES) into the substrate pathway, we were able to cause inhibition of transport that could be reversed with dithiothreitol. Importantly, co-incubation with PGE(2) protected PGT from this inhibition, suggesting that MTSES gains access to the aqueous pore pathway of PGT to form a mixed disulfide near the substrate-binding site. To identify the susceptible cysteine, we mutated, one at a time, all six of the putative transmembrane cysteines to serine. Only the mutation of Cys-530 to serine within putative transmembrane 10 became resistant to inhibition by MTSES. Thus, Cys-530 is the substrate-protectable, MTSES-inhibitable residue. To identify other residues that may be lining the substrate-binding site, we initiated cysteine-scanning mutagenesis of transmembrane 10 using Cys-530 as an entry point. On a C530S, MTSES-resistant background, residues in the N- and C-terminal directions were individually mutated to cysteine (Ala-513 to His-536), one at a time, and then analyzed for MTSES inhibition. Of the 24 cysteine-substituted mutants generated, 6 were MTSES-sensitive and, among these, 4 were substrate-protectable. The pattern of sensitivity to MTSES places these residues on the same face of an alpha-helix. The results of cysteine-scanning mutagenesis and molecular modeling of putative transmembrane 10 indicate that the substrate binding of PGT is formed among its membrane-spanning segments, with 4 residues along the cytoplasmic end of helix 10 contributing to one surface of the binding site.     

Label free quantitative proteomic analysis of secretome by Thermobifida fusca on different lignocellulosic biomass

Solid state fermentation of lignocellulosic biomass by filamentous microorganisms to induced enzyme production has been recognized as an attractive and cost effective technology. The secretion profile of lignocellulolytic enzymes by thermostable filamentous Thermobifida fusca (T. fusca) in solid state fermentation of different lignocellulosic biomasses, such as corn stover, hay; saw dust; sugarcane bagasse; wood chips; and un-dried green plant were explored using label-free exponentially modified protein abundance index (emPAI) based quantitative proteomics. Comparative analyses of T. fusca secretion profiles between cellulose and the various lignocellulosic biomasses showed induced expression of cellulolytic proteins by cellulose, and expression of hemicellulose, pectin and lignin degrading enzymes were induced by lignocellulosic biomasses. The solid state fermentation by T. fusca on lignocellulosic biomasses also revealed increased expressions of various transport proteins and hypothetical proteins. The Bray-Curtis similarity indices, clustering, and multidimensional scaling plot explicated differential protein expressions by T. fusca on different lignocellulosic biomasses, indicating that protein secretion by T. fusca is reliant on substrate complexity.     

The actin binding site of thymosin beta 4 mapped by mutational analysis.

We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin.