Editorial: Green chemistry biocatalysis

Editorial on the Research Topic: Green chemistry biocatalysi

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

© 2020, The Author(s). 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

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. <i>Methanococcus jannaschii</i> 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) <i>J. Bacteriol.</i>, <i>186</i>, 6360–6366]. Our structural studies show that the Thi4 orthologs from <i>M. jannaschii</i> and <i>Methanococcus igneus</i> are structurally similar to Thi4 from <i>Saccharomyces cerevisiae</i>. In addition, all active site residues are conserved except for a key cysteine residue, which in <i>S. cerevisiae</i> 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) <i>J. Am. Chem. Soc.</i>, DOI: 10.1021/jacs.6b00445]. Here we report X-ray crystal structures of Thi4 from <i>M. jannaschii</i> complexed with ADP-ribulose, the C205S variant of Thi4 from <i>S. cerevisiae</i> with a bound glycine imine intermediate, and Thi4 from <i>M. igneus</i> 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 <i>S</i> = 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 (<i>A</i>_iso = 0.0 ± 0.3 MHz) and dipolar couplings (<i>T</i> = 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 <i>A</i>_iso of 2.0 MHz and a <i>T</i> 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 <i>A</i>_iso of 0.0 MHz and a dipolar coupling (<i>T</i> = 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 <i>S</i> = 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 <i>S</i> = 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