Scopus İndeksli Yayınlar Koleksiyonu / Scopus Indexed Publications Collection
Permanent URI for this collectionhttps://hdl.handle.net/11147/7148
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Article Enhancing Electron Transfer in Cytochrome P450 Systems: Insights From CYP119-Putidaredoxin Interface Engineering(MDPI, 2025) Kakimova, Akbota; Surmeli, Nur BasakCytochrome P450 enzymes (CYPs) are versatile biocatalysts capable of performing selective oxidation reactions valuable for industrial and pharmaceutical applications. However, their catalytic efficiency is often constrained by dependence on costly electron donors, the requirement for redox partners, and uncoupling reactions that divert reducing power toward reactive oxygen species. Improving electron transfer efficiency through optimized redox partner interactions is therefore critical for developing effective CYP-based biocatalysts. In this study, we investigated the interaction between CYP119, a thermophilic CYP from Sulfolobus acidocaldarius, and putidaredoxin (Pdx), the redox partner of P450cam. Using rational design and computational modeling with PyRosetta 3, 14 CYP119 variants were modeled and analyzed by docking simulations on the Rosie Docking Server. Structural analysis identified three key mutations (N34E, D77R, and N34E/D77R) for site-directed mutagenesis. These mutations (N34E, D77R, and N34E/D77R) enhanced Pdx binding affinity by 20-, 3-, and 12-fold, respectively, without affecting substrate binding. Catalytic assays using lauric acid and indirect assays to monitor electron transfer revealed that, despite improved complex formation, the N34E variant showed reduced electron transfer efficiency compared to D77R. These findings highlight the delicate balance between redox partner binding affinity and catalytic turnover, emphasizing that fine-tuning electron transfer interfaces are essential for engineering efficient CYP biocatalysts.Article Citation - WoS: 13Citation - Scopus: 13Solid-Binding Peptide-Guided Spatially Directed Immobilization of Kinetically Matched Enzyme Cascades in Membrane Nanoreactors(American Chemical Society, 2021) Yücesoy, Deniz Tanıl; Akkineni, Susrut; Tamerler, Candan; Hinds, Bruce J.; Sarıkaya, MehmetBiocatalysis is a useful strategy for sustainable green synthesis of fine chemicals due to its high catalytic rate, reaction specificity, and operation under ambient conditions. Addressable immobilization of enzymes onto solid supports for one-pot multistep biocatalysis, however, remains a major challenge. In natural pathways, enzymes are spatially coupled to prevent side reactions, eradicate inhibitory products, and channel metabolites sequentially from one enzyme to another. Construction of a modular immobilization platform enabling spatially directed assembly of multiple biocatalysts would, therefore, not only allow the development of high-efficiency bioreactors but also provide novel synthetic routes for chemical synthesis. In this study, we developed a modular cascade flow reactor using a generalizable solid-binding peptide-directed immobilization strategy that allows selective immobilization of fusion enzymes on anodic aluminum oxide (AAO) monoliths with high positional precision. Here, the lactate dehydrogenase and formate dehydrogenase enzymes were fused with substrate-specific peptides to facilitate their self-immobilization through the membrane channels in cascade geometry. Using this cascade model, two-step biocatalytic production of l-lactate is demonstrated with concomitant regeneration of soluble nicotinamide adenine dinucleotide (NADH). Both fusion enzymes retained their catalytic activity upon immobilization, suggesting their optimal display on the support surface. The 85% cascading reaction efficiency was achieved at a flow rate that kinetically matches the residence time of the slowest enzyme. In addition, 84% of initial catalytic activity was preserved after 10 days of continuous operation at room temperature. The peptide-directed modular approach described herein is a highly effective strategy to control surface orientation, spatial localization, and loading of multiple enzymes on solid supports. The implications of this work provide insight for the single-step construction of high-power cascadic devices by enabling co-expression, purification, and immobilization of a variety of engineered fusion enzymes on patterned surfaces. © 2021 The Authors. Published by American Chemical Society.Article Citation - WoS: 3Citation - Scopus: 5A Novel Thermophilic Hemoprotein Scaffold for Rational Design of Biocatalysts(Springer Verlag, 2018) Efua Aggrey Fynn, Joana; Sürmeli, Nur BaşakHemoproteins are commonly found in nature, and involved in many important cellular processes such as oxygen transport, electron transfer, and catalysis. Rational design of hemoproteins can not only inspire novel biocatalysts but will also lead to a better understanding of structure-function relationships in native hemoproteins. Here, the heme nitric oxide/oxygen-binding protein from Caldanaerobacter subterraneus subsp. tengcongensis (TtH-NOX) is used as a novel scaffold for oxidation biocatalyst design. We show that signaling protein TtH-NOX can be reengineered to catalyze H2O2 decomposition and oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) by H2O2. In addition, the role of the distal tyrosine (Tyr140) in catalysis is investigated. The mutation of Tyr140 to alanine hinders the catalysis of the oxidation reactions. On the other hand, the mutation of Tyr140 to histidine, which is commonly observed in peroxidases, leads to a significant increase of the catalytic activity. Taken together, these results show that, while the distal histidine plays an important role in hemoprotein reactions with H2O2, it is not always essential for oxidation activity. We show that TtH-NOX protein can be used as an alternative scaffold for the design of novel biocatalysts with desired reactivity or functionality. H-NOX proteins are homologous to the nitric oxide sensor soluble guanylate cyclase. Here, we show that the gas sensor protein TtH-NOX shows limited capacity for catalysis of redox reactions and it can be used as a novel scaffold in biocatalysis design. [GRAPHICS] .Article Citation - WoS: 40Citation - Scopus: 50Hyaluronidase 1 and Ss-Hexosaminidase Have Redundant Functions in Hyaluronan and Chondroitin Sulfate Degradation(American Society for Biochemistry and Molecular Biology, 2012) Gushulak, Lara; Hemming, Richard; Martin, Dianna; Seyrantepe, Volkan; Pshezhetsky, Alexey; Triggs-Raine, BarbaraHyaluronan (HA), a member of the glycosaminoglycan (GAG) family, is a critical component of the extracellular matrix. A model for HA degradation that invokes the activity of both hyaluronidases and exoglycosidases has been advanced. However, no in vivo studies have been done to determine the extent to which these enzymes contribute to HA breakdown. Herein, we used mouse models to investigate the contributions of the endoglycosidase HYAL1 and the exoglycosidase β-hexosaminidase to the lysosomal degradation of HA. We employed histochemistry and fluorophore-assisted carbohydrate electrophoresis to determine the degree of HA accumulation in mice deficient in one or both enzyme activities. Global HA accumulation was present in mice deficient in both enzymes, with the highest levels found in the lymph node and liver. Chondroitin, a GAG similar in structure to HA, also broadly accumulated in mice deficient in both enzymes. Accumulation of chondroitin sulfate derivatives was detected in mice deficient in both enzymes, as well as in β-hexosaminidase-deficient mice, indicating that both enzymes play a significant role in chondroitin sulfate breakdown. Extensive accumulation of HA and chondroitin when both enzymes are lacking was not observed in mice deficient in only one of these enzymes, suggesting that HYAL1 and β-hexosaminidase are functionally redundant in HA and chondroitin breakdown. Furthermore, accumulation of sulfated chondroitin in tissues provides in vivo evidence that both HYAL1 and β-hexosaminidase cleave chondroitin sulfate, but it is a preferred substrate for β-hexosaminidase. These studies provide in vivo evidence to support and extend existing knowledge of GAG breakdown.Article Citation - WoS: 76Citation - Scopus: 90Optimization of a Growth Medium Using a Statistical Approach for the Production of an Alkaline Protease From a Newly Isolated Bacillus Sp. L21(Elsevier Ltd., 2006) Tarı, Canan; Gençkal, Hande; Tokatlı, FigenBacillus sp. L21 was isolated from the by-products of a leather factory (located in Izmir, Turkey) working under extreme alkaline conditions. Its phenotypic and genotypic identifications were completed, and determined as a potential alkaline protease producer. After screening various elements, carbon and nitrogen sources, soybean meal, maltose50, tween80 and the initial pH conditions were chosen as main factors to be used in the experimental design and response surface methodology (RSM) for the optimization of a low cost enzyme producing media for potential use on an industrial scale. The optimized values obtained by the statistical analysis showed that soybean meal at 3.0 g/l, maltose50 between the ranges of 30 and 40 g/l, tween80 at 0.35 g/l and an initial pH of 8.0 gives maximum protease activity.Article Citation - WoS: 56Citation - Scopus: 63Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance During S Phase(American Society for Biochemistry and Molecular Biology, 2006) Koç, Ahmet; Mathews, Christopher K.; Wheeler, Linda J.; Gross, Michael K.; Merrill, Gary FredericThioredoxin was initially identified by its ability to serve as an electron donor for ribonucleotide reductase in vitro. Whether it serves a similar function in vivo is unclear. In Saccharomyces cerevisiae, it was previously shown that Δtrx1 Δtrx2 mutants lacking the two genes for cytosolic thioredoxin have a slower growth rate because of a longer S phase, but the basis for S phase elongation was not identified. The hypothesis that S phase protraction was due to inefficient dNTP synthesis was investigated by measuring dNTP levels in asynchronous and synchronized wild-type and Δtrx1 Δtrx2 yeast. In contrast to wild-type cells, Δtrx1 Δtrx2 cells were unable to accumulate or maintain high levels of dNTPs when α-factor- or cdc15-arrested cells were allowed to reenter the cell cycle. At 80 min after release, when the fraction of cells in S phase was maximal, the dNTP pools in Δtrx1 Δtrx2 cells were 60% that of wild-type cells. The data suggest that, in the absence of thioredoxin, cells cannot support the high rate of dNTP synthesis required for efficient DNA synthesis during S phase. The results constitute in vivo evidence for thioredoxin being a physiologically relevant electron donor for ribonucleotide reductase during DNA precursor synthesis.Article Citation - WoS: 33Citation - Scopus: 37Relationship Between Morphology, Rheology and Polygalacturonase Production by Aspergillus Sojae Atcc 20235 in Submerged Cultures(Elsevier Ltd., 2006) Göğüş, Nihan; Tarı, Canan; Öncü, Şelale; Ünlütürk, Sevcan; Tokatlı, FigenA full factorial statistical design, with the factors of, two taxonomically different strains, seven types of seed culture formulations (slants) and two types of fermentation media were used to investigate the effect of these parameters on the morphology and polygalacturonase production. The rheology of the final fermentation medium was analyzed and appropriate mathematical model was applied to calculate suspension viscosity. It was found that most fermentation broths showed non-Newtonian flow behavior. According to statistical analysis, factors of strain types and fermentation media and the interaction between them were found significant on the enzyme activity. The effect of seed culture formulations (slants) were found insignificant at the significance level of 1%. Interaction of slants with strain types and fermentation media were also found insignificant. Considering the morphology of the final culture, Aspergillus sojae with the desired pellet morphology in a complex media, inoculated with a seed culture prepared from molasses resulted in maximum polygalacturonase enzyme activity (0.2 U/ml) and lowest suspension viscosity with a broth rheology close to Newtonian flow behavior.Article Citation - WoS: 4Citation - Scopus: 4Pentobarbital-Mediated Regulation of Alternative Polyadenylation in Drosophila Glutathione S-Transferase D21 Mrnas(American Society for Biochemistry and Molecular Biolog, 2004) Akgül, Bünyamin; Tu, Chen-Pei D.Two nearly identical, gstD21(L) and gstD21(S) mRNAs whose polyadenylation sites differ by 19 nucleotides, are transcribed from the intronless glutathione S-transferase D21 gene in Drosophila. Both mRNAs are intrinsically very labile, but exposure to pentobarbital renders them stabilized beyond what can be attributed to transcriptional activation. We have reconstituted this PB-mediated mRNA stabilization in a transgene (D21L) that contains the full-length gstD21(L) sequence. We have also constructed a similar transgene (D21L-UTR), which matches D21L but excluded the native 3′-UTR. D21L-UTR produces a relatively stable RNA, whose stability is unaffected by pentobarbital. Following pentobarbital treatment of wild-type flies, the levels of gstD21(L) and gstD21(S) mRNAs hold at a relatively constant ratio (L/S) of 1.4 ± 0.2. In transgenic flies, heat shock induction of D21L mRNA changed the L/S ratio to 0.6 ± 0.1, and it was further reduced to 0.3 ± 0.1 as D21L mRNA accumulated in the presence of PB. The ratio returned nearly normal (1.1 ± 0.1) as the D21L mRNA decayed over 12 h after terminating induction. In constrast, when D21L-UTR was present, the ratio remained constant (1.7 ± 0.2) even under various induction conditions and during recovery. Thus, the 3′-UTR, which was the critical difference between these two transgenes, must have some role in determining the L/S ratio. Induced D21L mRNA alone is not sufficient to cause reversible changes in the ratio. Such changes require the presence of pentobarbital. Therefore, pentobarbital may regulate this L/S ratio by affecting the choice of polyadenylation sites for the gstD21 mRNAs through sensing the concentrations of the native 3′-UTR sequences.Article Citation - WoS: 5Citation - Scopus: 5Evidence for a Stabilizer Element in the Untranslated Regions of Drosophila Glutathione S-Transferase D1 Mrna(American Society for Biochemistry and Molecular Biology Inc., 2002) Akgül, Bünyamin; Tu, Chen-Pei D.The neighboring genes gstD1 and gstD21 share 70% sequence identity, gstD1 encodes a 1,1,1-trichloro-2,2-bis-(P-chlorophenyl)ethane dehydrochlorinase; gstD21, a ligandin. Both of their mRNAs are inducible by pentobarbital but otherwise behave very differently. Intact gstD21 mRNA is intrinsically labile, but becomes stabilized when separated from its native untranslated region (UTR). In contrast, whereas gstD1 mRNA is very stable in its entirety, without its native UTRs it becomes even more labile than that of gstD21. Decay patterns from four chimeric D1-D21 mRNAs, designed to reveal the individual importance of each molecular region to stability, strongly indicate the presence of destabilizing elements in the coding region ofgstD1 mRNA. Thus, the UTRs of this molecule must contain a dominant stabilizer element that overrides the destabilizing influence of the coding region and confers overall stability to the entire molecule. The suspected presence of such a stabilizer element in gstD1 mRNA extends a concept from mRNA metabolism in yeast and cultured mammalian cells to include a multicellular organism, Drosophila melanogaster. The complementary presence of destabilizing and stabilizer elements on the same mRNA reveals a regulatory mechanism by which an abundant mRNA can be further induced by a chemical stimulus, or otherwise be returned to normal levels during recovery.