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BBA - Proteins and Proteomics (v.1834, #3)

Editorial Board (pp. i).

Influencing the monophenolase/diphenolase activity ratio in tyrosinase by Mor Goldfeder; Margarita Kanteev; Noam Adir; Ayelet Fishman (pp. 629-633).

Tyrosinase is a type 3 copper enzyme with great potential for production of commercially valuable diphenols from monophenols. However, the use of tyrosinase is limited by its further oxidation of diphenols to quinones. We recently determined the structure of the Bacillus megaterium tyrosinase revealing a residue, V218, which we proposed to take part in positioning of substrates within the active site. In the structure of catechol oxidase from Ipomoea batatas, the lack of monophenolase activity was attributed to the presence of F261 near CuA. Consequently, we engineered two variants, V218F and V218G. V218F was expected to have a decreased monophenolase activity, due to the bulky residue extending into the active site. Surprisingly, both V218F and V218G exhibited a 9- and 4.4-fold higher monophenolase/diphenolase activity ratio, respectively. X-ray structures of variant V218F display a flexibility of the phenylalanine residue along with an adjacent histidine, which we propose to be the source of the change in activity ratio.Display Omitted► A bulky residue positioned above CuA does not necessarily prevent tyrosinase hydroxylation onl-tyrosine. ► Tyrosinase V218 variants exhibit altered selectivity. ► Flexibility of H60 coordinating CuA affects the activity ratio of tyrosinase.

Keywords: Abbreviations; l; -Dopa; l; -3,4-dihydroxyphenylalanine; TyrBm; tyrosinase from; Bacillus megateriumTyrosinase; Bacillus megaterium; Rational design; Diphenol; Copper

A single mutation within a Ca2⁺ binding loop increases proteolytic activity, thermal stability, and surfactant stability by Mitsuyoshi Okuda; Tadahiro Ozawa; Masatoshi Tohata; Tsuyoshi Sato; Katsuhisa Saeki; Katsuya Ozaki (pp. 634-641).

We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca2⁺ ion. This residue lies 15–20Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate–enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca2⁺ and affects the packing of the Ca2⁺ binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.Display Omitted► KP-43 protease has a unique insertion (Asn187-Asp197), a part of Ca2⁺ binding loop. ► Tyr195Cys mutation increased both activity and stability of KP-43 protease. ► Tyr195Cys mutant enzyme showed 2.5-fold increased Ca2⁺ binding affinity. ► Molecular simulation showed high binding affinity was due to the thiolate of Cys195.

Keywords: Abbreviations; AAPL; N-glutaryl-; l; -Ala-; l; -Ala-; l; -Pro-; l; -Leu-; p; -nitroanilide; DTT; dithiothreitol; LAS; sodium linear alkylbenzene sulfate; 195C; Tyr195Cys mutantSubtilisin; Error-prone PCR; Proteolytic activity; Thermal stability; Surfactant stability; Calcium ion

Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein by Liliana Anjos; Ana S. Gomes; Eduardo P. Melo; Adelino V. Canário; Deborah M. Power (pp. 642-650).

Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca2⁺-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca2⁺ with high affinity (K _d=1.46nM) and favourable Gibbs free energy (∆ G=−12.4kcal/mol). The stoichiometry for Ca2⁺ bound to sbCRTAC2 at saturation indicated six Ca2⁺ ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca2⁺. Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell–cell and cell–matrix interactions.► CRTAC2 is mainly composed by β-sheet structures which favour its hyperthermostability. ► CRTAC2 binds Ca2⁺ with high affinity probably via integrin-like Ca2⁺-binding motifs. ► Protein aggregates of teleost CRTAC2 exist in vivo and in vitro. ► CRTAC2 promotes epithelial cell outgrowth in vitro.

Keywords: Recombinant Cartilage Acidic Protein 2; Protein folding; Soluble aggregate; Calcium; Teleost

Mutation of Val90 to His in the pseudoperoxidase from Leishmania major enhances peroxidase activity by Rina Saha; Moumita Bose; Subrata Adak (pp. 651-657).

Pseudoperoxidase from Leishmania major (LmPP) catalyzes the breakdown of peroxynitrite though it can hardly react with H₂O₂. Our modeling structure predicts that a conserved His to Val switch near the distal heme pocket of LmPP may determine the profile of its H₂O₂ activity. To test this hypothesis, we have generated complementary mutations in the LmPP (V90H) and studied the formation of Compounds I and II. The rate of transition from high spin ferric state of V90H to Compound I by H₂O₂ is increased by approximately three orders relative to wild-type LmPP, which is consistent with electron donor oxidation data where the V90H mutant enzyme is ~30 fold more active than wild type. Thus, our data indicate that a lower rate for heterolytic cleavage of the OO bond of H₂O₂ in wild type LmPP is caused by the His/Val switch in heme distal site. In the catalysis of peroxynitrite scavenging, V90H LmPP has lower catalytic activity compared to the wild type enzyme. In contrast to peroxynitrite scavenging, the second order rate constant of peroxynitrite binding step in mutant enzyme does not change significantly compared to the wild-type. Spectral data suggest that the distal Val90 residue in LmPP prevents the ferryl species formation in the presence of peroxynitrite. The lower peroxynitrite scavenging activity of the mutant reflects increased peroxidase activity rather than isomerase activity.► L. major pseudoperoxidase acts as a scavenger of peroxynitrite instead of H₂O₂. ► It contains an amino acid switch (His to Val) in heme distal site. ► The reactivity of V90H with H₂O₂ was increased ~2000 times. ► His/Val switch is important for altering enzymatic activity.

Keywords: Abbreviations; LmPP; Leishmania major; pseudoperoxidase; ABTS; 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); HRP; horseradish peroxidase; CCP; cytochrome c peroxidase; OONO; ⁻; peroxynitrite Leishmania; Heme protein; Peroxidase; Peroxynitrite; Steady-state catalysis; Rapid kinetics and mutation

The autocatalytic step is an integral part of the hydrogenase cycle by Banko Sarolta Bankó; Zsuzsanna Kucsma; Gábor Lente; Csaba Bagyinka (pp. 658-664).

We earlier proved the involvement of an autocatalytic step in the oxidation of H₂ by HynSL hydrogenase from Thiocapsa roseopersicina, and demonstrated that two enzyme forms interact in this step. Using a modified thin-layer reaction chamber which permits quantitative analysis of the concentration of the reaction product (reduced benzyl viologen) in the reaction volume during the oxidation of H₂, we now show that the steady-state concentration of the product displays a strong enzyme concentration dependence. This experimental fact can be explained only if the previously detected autocatalytic step occurs inside the catalytic enzyme-cycle and not in the enzyme activation process. Consequently, both interacting enzyme forms should participate in the catalytic cycle of the enzyme. As far as we are aware, this is the first experimental observation of such a phenomenon resulting in an apparent inhibition of the enzyme. It is additionally concluded that the interaction of the two enzyme forms should result in a conformational change in the enzyme–substrate form. This scheme is very similar to that of prion reactions. Since merely a few molecules are involved at some point of the reaction, this process is entirely stochastic in nature. We have therefore developed a stochastic calculation method, calculations with which lent support to the conclusion drawn from the experiment.Display Omitted► Steady-state in the hydrogenase reaction displays enzyme concentration dependence. ► This cannot be explained in the frame of a “normal” enzyme reaction. ► The autocatalytic step should be inside the enzyme cycle. ► Stochastic calculation method succeeded to describe the dependence.

Keywords: Hydrogenase; Autocatalysis; Enzyme kinetics; Kinetic model; Autocatalytic enzyme reaction; Autocatalytic extinction

Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level by Carlos Marcuello; Sonia Arilla-Luna; Milagros Medina; Anabel Lostao (pp. 665-676).

Biochemical characterization of Corynebacterium ammoniagenes FADS ( CaFADS) pointed to certain confusion about the stoichiometry of this bifunctional enzyme involved in the production of FMN and FAD in prokaryotes. Resolution of its crystal structure suggested that it might produce a hexameric ensemble formed by a dimer of trimers. We used atomic force microscopy (AFM) to direct imaging single CaFADS molecules bound to mica surfaces, while preserving their catalytic properties. AFM allowed solving individual CaFADS monomers, for which it was even possible to distinguish their sub-molecular individual N- and C-terminal modules in the elongated enzyme. Differences between monomers and higher stoichiometries were easily imaged, enabling us to detect formation of oligomeric species induced by ligand binding. The presence of ATP:Mg2⁺ particularly induced the appearance of the hexameric assembly whose mean molecular volume resembles the crystallographic dimer of trimers. Finally, the AFM results are confirmed in cross-linking solution, and the presence of such oligomeric CaFADS species detected in cell extracts. All these results are consistent with the formation of a dimer of trimers during the enzyme catalytic cycle that might bear biological relevance.Display Omitted► The mechanism of the bifunctional FAD synthetase from C. ammoniagenes was analyzed. ► Fluid AFM imaging determines different association patterns upon ligand binding. ► A dynamic transient formation of a compact dimer of trimers occurs during catalysis. ► Oligomeric FADS associations are proven within living cells. ► The assembly dependence on ligand binding must have a physiological significance.

Keywords: Abbreviations; FADS; FAD synthetase; RF; riboflavin; FMN; flavin mononucleotide; FAD; flavin adenine dinucleotide; ATP; adenosine 5′-triphosphate; RFK; ATP:riboflavin kinase; FMNAT; ATP:FMN adenylyltransferase; FADpp; FAD pyrophosphorylase; PIPES; 1,4-Piperazine diethane sulfonic acid; AFM; atomic force microscopy; UV; ultra-violet; SDS; sodium dodecyl-sulfate; BS; ₃; bis[sulfosuccinimidyl]suberate; BSOCOES; bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone; DTT; dithiothreitolFAD synthetase; Atomic force microscopy; Single-molecule; Macromolecular protein ensemble; Catalytic cycle

Off-pathway aggregation can inhibit fibrillation at high protein concentrations by Taru Deva; Nikolai Lorenzen; Brian S. Vad; Steen V. Petersen; Ida Thørgersen; Jan J. Enghild; Torsten Kristensen; Daniel E. Otzen (pp. 677-687).

Ribosomal protein S6 fibrillates readily at slightly elevated temperatures and acidic pH. We find that S6 fibrillation is retarded rather than favored when the protein concentration is increased above a threshold concentration of around 3.5mg/mL. We name this threshold concentration C_FR, the concentration at which fibrillation is retarded. Our data are consistent with a model in which this inhibition is due to the formation of an off-pathway oligomeric species with native-like secondary structure. The oligomeric species dominates at high protein concentrations but exists in dynamic equilibrium with the monomer so that seeding with fibrils can overrule oligomer formation and favors fibrillation under C_FR conditions. Thus, fibrillation competes with formation of off-pathway oligomers, probably due to a monomeric conversion step that is required to commit the protein to the fibrillation pathway. The S6 oligomer is resistant to pepsin digestion. We also report that S6 forms different types of fibrils dependent on protein concentration. Our observations highlight the multitude of conformational states available to proteins under destabilizing conditions.Display Omitted► Fibrillation of protein S6 is retarded above a certain threshold concentration ( C_FR). ► C_FR mediates fibril inhibition via a soluble, native-like, off-pathway intermediate. ► Equilibrium shift between monomer and off-pathway intermediate is important. ► Fibril morphology is dependent on protein concentration.

Keywords: Abbreviations; AFM; atomic force microscopy; ATR-FTIR; attenuated total reflectance-Fourier transform infrared spectroscopy; CAC; critical aggregation concentration; CD; circular dichroism; C; _FR; concentration at which fibrillation is retarded; DLS; dynamic light scattering; IPTG; isopropyl β-; d; -thiogalactopyranoside; PAGE; polyacrylamide gel electrophoresis; PCR; polymerase chain reaction; PVDF; polyvinylidene fluoride; SDS; sodium dodecyl sulfate; SCC; super-critical concentration; ThT; thioflavin T; Tris; tris(hydroxymethyl)aminomethaneAmyloid formation; S6; Soluble oligomers; Proteolysis; Fibril morphology; Acid cleavage

Cytochrome P450_cin (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s by Jeanette E. Stok; Sean Yamada; Anthony J. Farlow; Kate E. Slessor; James J. De Voss (pp. 688-696).

P450_cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450_cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450_cin D241N mutant has been produced and found to be analogous to the P450_cam D251N mutant. P450_cin catalyses the hydroxylation of cineole to give only (1 R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450_cin D241N mutant also hydroxylated cineole to produce only (1 R)-6β-hydroxycineole, was moderately well coupled (31±3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450_cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450_cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase.► P450_cin D241N has similar biochemical properties to P450_cam D251N. ► D241N is moderately well coupled but oxidises cineole at 2% of wild type rate. ► No ferric-peroxo chemistry was observed with a variety of substrates. ► Products from D241N was the same as for the wildtype with a range of substrates.

Keywords: Cytochrome P450; Cineole; Citrobacter braakii; Enzyme mechanism

Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator by M.A.S. Toledo; C.A. Santos; J.S. Mendes; A.C. Pelloso; L.L. Beloti; A. Crucello; M.T.P. Favaro; A.S. Santiago; D.R.S. Schneider; A.M. Saraiva; D.R. Stach-Machado; A.A. Souza; D.B.B. Trivella; R. Aparicio; L. Tasic; A.R. Azzoni; A.P. Souza (pp. 697-707).

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis, cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.Display Omitted► Recombinant XfLysRL was successfully expressed and purified in Escherichia coli. ► SAXS envelope and in silico model for XfLysRL were obtained. ► XfLysRL dimer has similar structure to Acinetobacter baylyi BenM dimer. ► XfLysRL is able to interact with muconate and benzoate, co-inducers of BenM. ► XfLysRL is differentially expressed during biofilm and planktonic growth.

Keywords: LysR-like transcriptional regulator (LTTR); Small-angle X-ray scattering (SAXS); Benzoate; Muconate; Biofilm; Citrus variegated chlorosis (CVC)

Polyproline fold—In imparting kinetic stability to an alkaline serine endopeptidase by Sonali B. Rohamare; Vaishali Dixit; Pavan Kumar Nareddy; D. Sivaramakrishna; Musti J. Swamy; Sushama M. Gaikwad (pp. 708-716).

Polyproline II (PPII) fold, an unusual structural element was detected in the serine protease from Nocardiopsis sp. NCIM 5124 (NprotI) based on far UV circular dichroism spectrum, structural transitions of the enzyme in presence of GdnHCl and a distinct isodichroic point in chemical and thermal denaturation. The functional activity and conformational transitions of the enzyme were studied under various denaturing conditions. Enzymatic activity of NprotI was stable in the vicinity of GdnHCl upto 6.0M concentration, organic solvents viz. methanol, ethanol, propanol (all 90% v/v), acetonitrile (75% v/v) and proteases such as trypsin, chymotrypsin and proteinase K (NprotI:protease 10:1). NprotI seems to be a kinetically stable protease with a high energy barrier between folded and unfolded states. Also, an enhancement in the activity of the enzyme was observed in 1M GdnHCl upto 8h, in organic solvents (75% v/v) for 72h and in presence of proteolytic enzymes. The polyproline fold remained unaltered or became more prominent under the above mentioned conditions. However, it diminished gradually during thermal denaturation above 60°C. Thermal transition studies by differential scanning calorimetry (DSC) showed scan rate dependence as well as irreversibility of denaturation, the properties characteristic of kinetically stable proteins. This is the first report of PPII helix being the global conformation of a non structural protein, an alkaline serine protease, from a microbial source, imparting kinetic stability to the protein.► Detection of polyproline (PPII) fold in an alkaline serine endopeptidase ► Unusual stability of the enzyme in 6M GdnHCl ► Activity enhancement in 90% ( v/v) alcohols and in presence of proteases ► Kinetically controlled unfolding of the protein as studied by DSC ► Role of PPII fold in imparting kinetic stability

Keywords: Nocardiopsis; sp.; Serine protease; Polyproline fold; Conformation; Kinetic stability; Differential scanning calorimetry

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BBA - Proteins and Proteomics (v.1834, #3)