Spectroscopic and Kinetic Investigation of the Catalytic Mechanism of Tyrosine Hydroxylase

Tyrosine Hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme iron oxygenase. TyrH catalyzes the hydroxylation reaction of tyrosine to dihydroxyphenylalanine (DOPA). This reaction is the first and the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. The active site iron in TyrH is coordinated by the common facial triad motif, 2-His-1-Glu. A combination of kinetic and spectroscopic techniques was applied in order to obtain insight into the catalytic mechanism of this physiologically important enzyme. Analysis of the TyrH reaction by rapid freeze-quench Mossbauer spectroscopy allowed the first direct characterization of an Fe(IV) intermediate in a mononuclear nonheme enzyme catalyzing aromatic hydroxylation. Further rapid kinetic studies established the kinetic competency of this intermediate to be the long-postulated hydroxylating species, Fe(IV)O. Spectroscopic investigations of wild-type (WT) and mutant TyrH complexes using magnetic circular dichroism (MCD) and X-ray absorption spectroscopy (XAS) showed that the active site iron is 6-coordinate in the resting form of the enzyme and that binding of either tyrosine or 6MPH4 alone does not change the coordination. However, when both tyrosine and 6MPH4 are bound, the active site becomes 5-coordinate, creating an open site for reaction with O2. Investigation of the kinetics of oxygen reactivity of TyrH complexes in the absence and presence of tyrosine and/or 6MPH4 indicated that there is a significant enhancement in reactivity in the 5-coordinate complex in comparison to the 6-coordinate form. Similar investigations with E332A TyrH showed that Glu332 residue plays a role in directing the protonation of the bridged complex that forms prior to the formation of Fe(IV)O. Rapid chemical quench analyses of DOPA formation showed a burst of product formation, suggesting a slow product release step. Steady-state viscosity experiments established a diffusional step as being significantly rate-limiting. Further studies with stopped-flow spectroscopy indicated that the rate of TyrH reaction is determined by a combination of a number of physical and chemical steps. Investigation of the NO complexes of TyrH by means of optical absorption, electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) techniques revealed the relative positions of the substrate and cofactor with respect to NO, an O2 mimic, and provided further insight into how the active site is tuned for catalytic reactivity upon substrate and cofactor binding

A Bi-Enzymatic Cascade Pathway Towards FAHFAs

Recently discovered endogenous mammalian lipids fatty acid esters of hydroxy fatty acids (FAHFAs), proved to have anti-inflammatory and anti-diabetic effects. Due to their extremely low abundancies in vivo, forging a feasible scenario for FAHFA synthesis is critical for their use in uncovering biological mechanism or clinical trials. Here, we showcase a fully enzymatic approach, a novel in vitro bi-enzymatic cascade system, enabling an effective conversion of nature-abundant fatty acid into FAHFAs. Two hydratases from L. acidophilus were used for converting unsaturated fatty acids to various stereospecific hydroxy fatty acids, followed by esterification with another fatty acid catalyzed by C. antarctica lipase A (CALA). Various FAHFAs were synthesized in a preparative scale using this bi-enzymatic approach in a one-pot two-step operation mode. In all, we demonstrated that hydratase-CALA system promises a sustainable solution to the synthesis of structure-diverse stereospecific FAHFAs

Theoretical study on the glycosidic C–C bond cleavage of 3’’-oxo-puerarin

Abstract Puerarin, daidzein C-glucoside, was known to be biotransformed to daidzein by human intestinal bacteria, which is eventually converted to (S)-equol. The metabolic pathway of puerarin to daidzein by DgpABC of Dorea sp. PUE strain was reported as puerarin (1) → 3’’-oxo-puerarin (2) → daidzein (3) + hexose enediolone (C). The second reaction is the cleavage of the glycosidic C–C bond, supposedly through the quinoid intermediate (4). In this work, the glycosidic C–C bond cleavage reaction of 3’’-oxo-puerarin (2) was theoretically studied by means of DFT calculation to elucidate chemical reaction mechanism, along with biochemical energetics of puerarin metabolism. It was found that bioenergetics of puerarin metabolism is slightly endergonic by 4.99 kcal/mol, mainly due to the reaction step of hexose enediolone (C) to 3’’-oxo-glucose (A). The result implied that there could be additional biochemical reactions for the metabolism of hexose enediolone (C) to overcome the thermodynamic energy barrier of 4.59 kcal/mol. The computational study focused on the C–C bond cleavage of 3’’-oxo-puerarin (2) found that formation of the quinoid intermediate (4) was not accessible thermodynamically, rather the reaction was initiated by the deprotonation of 2’’C–H proton of 3’’-oxo-puerarin (2). The 2’’C-dehydro-3’’-oxo-puerarin (2a2C) anionic species produced hexose enediolone (C) and 8-dehydro-daidzein anion (3a8), and the latter quickly converted to daidzein through the daidzein anion (3a7). Our study also explains why the reverse reaction of C-glycoside formation from daidzein (3) and hexose enediolone (C) is not feasible

Diabetes Mellitus and Epigenetic Mechanisms

Diabetes Mellitus (DM) is an important disease caused by insulin deficiency or insulin receptor resistance and characterized by hyperglycemia. The prevalence rate of DM is increasing rapidly worldwide and its associated complications affect the quality of life of patients adverse­ly. In addition, high medical costs for its treatment bring significant economic load on countries. Epigenetics is the reversible modifications on the genome, which lead to changes in gene expression without any alteration in the DNA sequence. Epigenetic modifications can easily be affected by environmental factors and abnormalities in these modifications have been linked to many diseases including cancer and neurodegenerative disorders. In this review, we will summarize the relationship of DM and its complications with DNA and RNA methylation, which are among the most important modifications

Optimization and Engineering of a Self-Sufficient CYP102 Enzyme from Bacillus amyloliquefaciens towards Synthesis of In-Chain Hydroxy Fatty Acids

Cytochrome P450 (CYP) mediated enzymatic hydroxylation of fatty acids present a green alternative to chemical synthesis of hydroxy fatty acids (HFAs), which are high-value oleochemicals with various uses in materials industry and medical field. Although many CYPs require the presence of additional reductase proteins for catalytic activity, self-sufficient CYPs have their reductase partner naturally fused into their catalytic domain, leading to a greatly simplified biotransformation process. A recently discovered self-sufficient CYP, BAMF2522 from Bacillus amyloliquefaciens DSM 7, exhibits novel regioselectivity by hydroxylating in-chain positions of palmitic acid generating ω-1 to ω-7 HFAs, a rare regiodiversity profile among CYPs. Besides, F89I mutant of BAMF2522 expanded hydroxylation up to ω-9 position of palmitic acid. Here, we further characterize this enzyme by determining optimum temperature and pH as well as thermal stability. Moreover, using extensive site-directed and site-saturation mutagenesis, we obtained BAMF2522 variants that demonstrate greatly increased regioselectivity for in-chain positions (ω-4 to ω-9) of various medium to long chain fatty acids. Remarkably, when a six-residue mutant was reacted with palmitic acid, 84% of total product content was the sum of ω-7, ω-8 and ω-9 HFA products, the highest in-chain selectivity observed to date with a self-sufficient CYP. In short, our study demonstrates the potential of a recently identified CYP and its mutants for green and sustainable production of a variety of in-chain hydroxy enriched HFAs

Biocatalytic conversion of fatty acids into drop-in biofuels: Towards sustainable energy sources

Intensifying effects of global warming necessitates swift solutions for decarbonization. A big source for the increasing atmospheric CO2 is burning of transportation fuels. Although electrification of light vehicles is an important step to reduce CO2 emissions, carbon-based fuels will still be likely to power up heavy-duty transport, marine transport, and aviation for some time. Thus, the fastest way to decarbonize these sectors could be an immediate transition to carbon-neutral biofuels. Recently, conversion of fatty acids into hydrocarbons, as “drop-in biofuels”, is emerging as a promising alternative to fossil fuels. However, current technologies rely on energy intensive chemical methods that are not quite eco-friendly. Therefore, enzymatic conversion of fatty acids into hydrocarbons present a benign production method. In the last decade, multiple enzymes have been discovered to convert free fatty acids into alka(e)nes. Here, we introduce these enzymes with a description of the studies performed so far, especially regarding cell-free biocatalysis

Relationship between fecal calprotectin level and disease activity in patients with hidradenitis suppurativa

Background Hidradenitis suppurativa is a chronic, inflammatory, recurrent disease with recurrent abscesses, and sinus tract formation leading to scarring. Calprotectin has immunomodulatory, antimicrobial, and antiproliferative properties and is a calcium-binding protein primarily found in the neutrophil cytoplasm. In recent years, a significant relationship between the activity of various diseases and the level of calprotectin has led to the conclusion that there may be a similar relationship in hidradenitis suppurativa

Rational Engineering of Hydratase from Lactobacillus Acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

Enzymatic conversion of abundant fatty acids (FAs) through fatty acid hydratases (FAHs) presents an environment-friendly and efficient route for production of high-value hydroxy fatty acids (HFAs). However, a limited diversity was achieved among HFAs to date with respect to chain length and hydroxy group position, due to high substrate- and regio-selectivity of hydratases. In this study, we compared two highly similar FAHs from Lactobacillus acidophilus: FA-HY2 has narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilize longer chain substrates and hydrate various double bond positions. We reveal three active-site residues that play remarkable role in directing substrate specificity and regioselectivity of hydration. When these residues on FA-HY2 are mutated to the corresponding residues in FA-HY1, we observe a significant expansion of substrate scope and distinct shift and enhancement in hydration of double bonds towards omega-end of FAs. A three-residue mutant of FA-HY2 (TM-FA-HY2; T391S/H393S/I378P) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting ratio of the HFA product regioisomers (10-OH:13-OH) from 99:1 to 12:88. Although kcat values are still low in comparison to wild-type FA-HY1, TM-FA-HY2 exhibited about 60-fold increase in catalytic efficiency (kcat/Km) compared to wild-type FA-HY2. Important changes in regioselectivity were also observed with mutant enzymes for arachidonic acid and C18 PUFAs. In addition, TM-FA-HY2 variant exhibited high conversion rates for cis-5, cis-8, cis-11, cis-14, cis-17-eicosapentaenoic acid (EPA) and cis-8, cis-11, cis-14-eicosatrienoic acid (ETA) at preparative scale and enabled isolation of 12-hydroxy products with moderate yields. Furthermore, we demonstrated the potential of microalgae as a source of diverse FAs for HFA production. Our study paves the way for tailor-made FAH design and for efficient conversion of FA sources into diverse range of HFAs with high potential for various applications from polymer industry to medical field.</div