Synthesis of high-titer alka(e)nes in Yarrowia lipolytica is enabled by a discovered mechanism.

Alka(e)nes are ideal fuel components for aviation, long-distance transport, and shipping. They are typically derived from fossil fuels and accounting for 24% of difficult-to-eliminate greenhouse gas emissions. The synthesis of alka(e)nes in Yarrowia lipolytica from CO2-neutral feedstocks represents an attractive alternative. Here we report that the high-titer synthesis of alka(e)nes in Yarrowia lipolytica harboring a fatty acid photodecarboxylase (CvFAP) is enabled by a discovered pathway. We find that acyl-CoAs, rather than free fatty acids (FFAs), are the preferred substrate for CvFAP. This finding allows us to debottleneck the pathway and optimize fermentation conditions so that we are able to redirect 89% of acyl-CoAs from the synthesis of neutral lipids to alka(e)nes and reach titers of 1.47 g/L from glucose. Two other CO2-derived substrates, wheat straw and acetate, are also demonstrated to be effective in producing alka(e)nes. Overall, our technology could advance net-zero emissions by providing CO2-neutral and energy-dense liquid biofuels.

Gut Metabolism of Furanocoumarins: Proposed Function of Co O-Methyltransferase.

Gut metabolism of natural products is of great interest due to the altered biological activity of the metabolites. To study the gut metabolism of the dietary furanocoumarins, the biotransformation of Angelica dahurica was studied with human gut microbiota. The major components of Avenula dahurica, including xanthotoxin (1), bergapten (2), imperatorin (3), isoimperatorin (4), oxypeucedanin (5), and byakangelicol (6), were all metabolized by the human fecal sample, and each furanocoumarin was also biotransformed by Blautia sp. MRG-PMF1 responsible for intestinal O-demethylation. Oxypeucedanin (5) and byakangelicol (6) were converted to oxypeucedanin hydrate (9) and desmethylbyakangelicin (12), respectively. The gut microbial conversion of xanthotoxin (1) and bergapten (2) with the MRG-PMF1 strain resulted in the production of xanthotoxol (7) and bergaptol (8), respectively, due to the methyl aryl ether cleavage by O-methyltransferase. Unexpectedly, the biotransformation of prenylated furanocoumarins, imperatorin (3), and isoimperatorin (4) resulted in the corresponding deprenylated furanocoumarins of xanthotoxol (7) and bergaptol (8), respectively. The cleavage of the prenyl aryl ether group by gut microbiota was unprecedented metabolism. Our data presented the first deprenylation of prenylated natural products, presumably by the anaerobic prenyl aryl ether cleavage reaction catalyzed by Co-corrinoid enzyme.

From Suicide Enzyme to Catalyst: The Iron-Dependent Sulfide Transfer in Methanococcus jannaschii Thiamin Thiazole Biosynthesis.

Bacteria and yeast utilize different strategies for sulfur incorporation in the biosynthesis of the thiamin thiazole. Bacteria use thiocarboxylated proteins. In contrast, Saccharomyces cerevisiae thiazole synthase (THI4p) uses an active site cysteine as the sulfide source and is inactivated after a single turnover. Here, we demonstrate that the Thi4 ortholog from Methanococcus jannaschii uses exogenous sulfide and is catalytic. Structural and biochemical studies on this enzyme elucidate the mechanistic details of the sulfide transfer reactions.

Structural Basis for Iron-Mediated Sulfur Transfer in Archael and Yeast Thiazole Synthases.

Thiamin diphosphate is an essential cofactor in all forms of life and plays a key role in amino acid and carbohydrate metabolism. Its biosynthesis involves separate syntheses of the pyrimidine and thiazole moieties, which are then coupled to form thiamin monophosphate. A final phosphorylation produces the active form of the cofactor. In most bacteria, six gene products are required for biosynthesis of the thiamin thiazole. In yeast and fungi only one gene product, Thi4, is required for thiazole biosynthesis. Methanococcus jannaschii expresses a putative Thi4 ortholog that was previously reported to be a ribulose 1,5-bisphosphate synthase [Finn, M. W. and Tabita, F. R. (2004) J. Bacteriol., 186, 6360-6366]. Our structural studies show that the Thi4 orthologs from M. jannaschii and Methanococcus igneus are structurally similar to Thi4 from Saccharomyces cerevisiae. In addition, all active site residues are conserved except for a key cysteine residue, which in S. cerevisiae is the source of the thiazole sulfur atom. Our recent biochemical studies showed that the archael Thi4 orthologs use nicotinamide adenine dinucleotide, glycine, and free sulfide to form the thiamin thiazole in an iron-dependent reaction [Eser, B., Zhang, X., Chanani, P. K., Begley, T. P., and Ealick, S. E. (2016) J. Am. Chem. Soc. , DOI: 10.1021/jacs.6b00445]. Here we report X-ray crystal structures of Thi4 from M. jannaschii complexed with ADP-ribulose, the C205S variant of Thi4 from S. cerevisiae with a bound glycine imine intermediate, and Thi4 from M. igneus with bound glycine imine intermediate and iron. These studies reveal the structural basis for the iron-dependent mechanism of sulfur transfer in archael and yeast thiazole synthases.

HYSCORE Analysis of the Effects of Substrates on Coordination of Water to the Active Site Iron in Tyrosine Hydroxylase.

Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O2 at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O2 to poise the active site iron in an S = ³/2 {FeNO}7 form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H2O molecule characterized by proton isotropic hyperfine couplings (A(iso) = 0.0 ± 0.3 MHz) and dipolar couplings (T = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an A(iso) of 2.0 MHz and a T of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H2O bound to {FeNO}7 model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH4, the HYSCORE cross peaks attributed to H2O and OH⁻ for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an A(iso) of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N5 proton of the reduced pterin.

Pulsed EPR study of amino acid and tetrahydropterin binding in a tyrosine hydroxylase nitric oxide complex: evidence for substrate rearrangements in the formation of the oxygen-reactive complex.

Tyrosine hydroxylase is a nonheme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin, and molecular oxygen. We have used nitric oxide as an O2 surrogate to poise Fe(II) at the catalytic site in an S = 3/2, {FeNO}7 form amenable to EPR spectroscopy. ²H-electron spin echo envelope modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}7 paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}7 center to the closest deuteron on 6,7-²H-6-methyltetrahydropterin from >5.9 Å to 4.4 ± 0.2 Å. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-²H-tyrosine resulted in reorientation of the magnetic axes of the S = 3/2, {FeNO}7 center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase.

Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor.

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen [Das et al. (2011) Angew. Chem. 50, 7148-7152]. Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions and that the oxygen in formate derives from water. The very low activity (<1 turnover/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turnover, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, k(prod) = 3.4 ± 0.5 min(-1), is observed, indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, k(cat) = 0.17 ± 0.01 min(-1) and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.

Measurement of intrinsic rate constants in the tyrosine hydroxylase reaction.

Tyrosine hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme aromatic amino acid hydroxylase that catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA). Chemical quench analyses of the enzymatic reaction show a burst of DOPA formation, followed by a linear rate equal to the k(cat) value at both 5 and 30 degrees C. The effects of increasing solvent viscosity confirm that k(cat) is approximately 84% limited by diffusion, most probably due to slow product release, and that tyrosine has a commitment to catalysis of 0.45. The effect of viscosity on the k(cat)/K(m) for 6-methyltetrahydropterin is greater than the theoretical limit, consistent with the coupling of pterin binding to the movement of a surface loop. The absorbance changes in the spectrum of the tetrahydropterin during the first turnover, the kinetics of DOPA formation during the first turnover, and the previously described kinetics for formation and decay of the Fe(IV)O intermediate [Eser, B. E., Barr, E. W., Frantom, P. A., Saleh, L., Bollinger, J. M., Jr., Krebs, C., and Fitzpatrick, P. F. (2007) J. Am. Chem. Soc. 129, 11334-11335] were analyzed globally, yielding a single set of rate constants for the TyrH reaction. Reversible binding of oxygen is followed by formation of Fe(IV)O and 4a-hydroxypterin with a rate constant of 13 s(-1) at 5 degrees C. Transfer of oxygen from Fe(IV)O to tyrosine to form DOPA follows with a rate constant of 22 s(-1). Release of DOPA and/or the 4a-hydroxypterin with a rate constant of 0.86 s(-1) completes the turnover.

Spectroscopy and kinetics of wild-type and mutant tyrosine hydroxylase: mechanistic insight into O2 activation.

Tyrosine hydroxylase (TH) is a pterin-dependent nonheme iron enzyme that catalyzes the hydroxylation of L-tyr to L-DOPA in the rate-limiting step of catecholamine neurotransmitter biosynthesis. We have previously shown that the Fe(II) site in phenylalanine hydroxylase (PAH) converts from six-coordinate (6C) to five-coordinate (5C) only when both substrate + cofactor are bound. However, steady-state kinetics indicate that TH has a different co-substrate binding sequence (pterin + O(2) + L-tyr) than PAH (L-phe + pterin + O(2)). Using X-ray absorption spectroscopy (XAS), and variable-temperature-variable-field magnetic circular dichroism (VTVH MCD) spectroscopy, we have investigated the geometric and electronic structure of the wild-type (WT) TH and two mutants, S395A and E332A, and their interactions with substrates. All three forms of TH undergo 6C --> 5C conversion with tyr + pterin, consistent with the general mechanistic strategy established for O(2)-activating nonheme iron enzymes. We have also applied single-turnover kinetic experiments with spectroscopic data to evaluate the mechanism of the O(2) and pterin reactions in TH. When the Fe(II) site is 6C, the two-electron reduction of O(2) to peroxide by Fe(II) and pterin is favored over individual one-electron reactions, demonstrating that both a 5C Fe(II) and a redox-active pterin are required for coupled O(2) reaction. When the Fe(II) is 5C, the O(2) reaction is accelerated by at least 2 orders of magnitude. Comparison of the kinetics of WT TH, which produces Fe(IV)=O + 4a-OH-pterin, and E332A TH, which does not, shows that the E332 residue plays an important role in directing the protonation of the bridged Fe(II)-OO-pterin intermediate in WT to productively form Fe(IV)=O, which is responsible for hydroxylating L-tyr to L-DOPA.

Liquid crystalline mesophases of pluronics (L64, P65, and P123) and transition metal nitrate salts ([M(H2O)6](NO3)2).

The triblock poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, Pluronics (L64, P65, and P123), form liquid crystalline (LC) mesophases with transition metal nitrate salts (TMS), [M(H(2)O)(n)](NO(3))(2), in the presence and absence of free water in the media. In this assembly process, M-OH(2) plays an important role as observed in a TMS:C(n)EO(m) (C(n)EO(m) is oligo(ethylene oxide) nonionic surfactants) system. The structure of the LC mesophases and interactions of the metal ion-nitrate ion and metal ion-Pluronic were investigated using microscopy (POM), diffraction (XRD), and spectroscopy (FTIR and micro-Raman) techniques. The TMS:L64 system requires a shear force for mesophase ordering to be observed using X-ray diffraction. However, TMS:P65 and TMS:P123 form well structured LC mesophases. Depending on the salt/Pluronic mole ratio, hexagonal LC mesophases are observed in the TMS:P65 systems and cubic and tetragonal LC mesophases in the TMS:P123 systems. The LC mesophase in the water/salt/Pluronic system is sensitive to the concentration of free (H(2)O) and coordinated water (M-OH(2)) molecules and demonstrates structural changes. As the free water is evaporated from the H(2)O:TMS:Pluronic LC mesophase (ternary mixture), the nitrate ion remains free in the media. However, complete evaporation of the free water molecules enforces the coordination of the nitrate ion to the metal ion in all TMS:Pluronic systems.