Increasing lysergic acid levels for ergot alkaloid biosynthesis: Directing catalysis via the F-G loop of Clavine oxidases.

Most ergot alkaloid drugs are semi-synthetically derived from the natural product lysergic acid, a valuable precursor for the development of novel ergot alkaloid drugs. Clavine oxidase (CloA) is a putative cytochrome P450, identified in the ergot alkaloid biosynthesis pathway, and a key enzyme that catalyzes the formation of lysergic acid from the precursor alkaloid agroclavine in a two-step oxidation reaction. We demonstrated in this study that Saccharomyces cerevisiae can be used as a viable host for the functional expression of CloA from Claviceps purpurea and its orthologs. We also showed that CloA orthologs differ in their ability to oxidize the substrate agroclavine, with some orthologs only able to perform the first oxidation reaction to produce elymoclavine. Of particular note, we identified a region between the F-G helices of the enzyme that may be involved in directing oxidation of agroclavine by substrate recognition and uptake. Using this knowledge, engineered CloAs were shown to produce lysergic acid at levels exceeding that of wildtype CloA orthologs; a CloA variant, chimeric AT5 9Hypo CloA, increased production levels of lysergic acid to 15 times higher as compared to the wildtype enzyme, demonstrating future utility for the industrial production of ergot alkaloids using biosynthetic routes.

A high-throughput pipeline for scalable kit-free RNA extraction.

An overreliance on commercial, kit-based RNA extraction in the molecular diagnoses of infectious disease presents a challenge in the event of supply chain disruptions and can potentially hinder testing capacity in times of need. In this study, we adapted a well-established, robust TRIzol-based RNA extraction protocol into a high-throughput format through miniaturization and automation. The workflow was validated by RT-qPCR assay for SARS-CoV-2 detection to illustrate its scalability without interference to downstream diagnostic sensitivity and accuracy. This semi-automated, kit-free approach offers a versatile alternative to prevailing integrated solid-phase RNA extraction proprietary systems, with the added advantage of improved cost-effectiveness for high volume acquisition of quality RNA whether for use in clinical diagnoses or for diverse molecular applications.

Biosynthesis of Nature-Inspired Unnatural Cannabinoids.

Natural products make up a large proportion of medicine available today. Cannabinoids from the plant Cannabis sativa is one unique class of meroterpenoids that have shown a wide range of bioactivities and recently seen significant developments in their status as therapeutic agents for various indications. Their complex chemical structures make it difficult to chemically synthesize them in efficient yields. Synthetic biology has presented a solution to this through metabolic engineering in heterologous hosts. Through genetic manipulation, rare phytocannabinoids that are produced in low yields in the plant can now be synthesized in larger quantities for therapeutic and commercial use. Additionally, an exciting avenue of exploring new chemical spaces is made available as novel derivatized compounds can be produced and investigated for their bioactivities. In this review, we summarized the biosynthetic pathways of phytocannabinoids and synthetic biology efforts in producing them in heterologous hosts. Detailed mechanistic insights are discussed in each part of the pathway in order to explore strategies for creating novel cannabinoids. Lastly, we discussed studies conducted on biological targets such as CB1, CB2 and orphan receptors along with their affinities to these cannabinoid ligands with a view to inform upstream diversification efforts.

Synthetic Enzymology and the Fountain of Youth: Repurposing Biology for Longevity.

Caloric restriction (CR) is an intervention that can increase maximal lifespan in organisms, but its application to humans remains challenging. A more feasible approach to achieve lifespan extension is to develop CR mimetics that target biochemical pathways affected by CR. Recent studies in the engineering and structural characterization of polyketide synthases (PKSs) have facilitated their use as biocatalysts to produce novel polyketides. Here, we show that by establishing a combinatorial biosynthetic route in Escherichia coli and exploring the substrate promiscuity of a mutant PKS from alfalfa, 413 potential anti-ageing polyketides were biosynthesized. In this approach, novel acyl-coenzyme A (CoA) precursors generated by promiscuous acid-CoA ligases were utilized by PKS to generate polyketides which were then fed to Caenorhabditis elegans to study their potential efficacy in lifespan extension. It was found that CR mimetics like resveratrol can counter the age-associated decline in mitochondrial function and increase the lifespan of C. elegans. Using the mitochondrial respiration profile of C. elegans supplemented for 8 days with 50 µM resveratrol as a blueprint, we can screen our novel polyketides for potential CR mimetics with improved potency. This study highlights the utility of synthetic enzymology in the development of novel anti-ageing therapeutics.

Exploiting the Biosynthetic Potential of Type III Polyketide Synthases.

Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs) using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development.

Enzyme architecture: the effect of replacement and deletion mutations of loop 6 on catalysis by triosephosphate isomerase.

Two mutations of the phosphodianion gripper loop in chicken muscle triosephosphate isomerase (cTIM) were examined: (1) the loop deletion mutant (LDM) formed by removal of residues 170-173 [Pompliano, D. L., et al. (1990) Biochemistry 29, 3186-3194] and (2) the loop 6 replacement mutant (L6RM), in which the N-terminal hinge sequence of TIM from eukaryotes, 166-PXW-168 (X = L or V), is replaced by the sequence from archaea, 166-PPE-168. The X-ray crystal structure of the L6RM shows a large displacement of the side chain of E168 from that for W168 in wild-type cTIM. Solution nuclear magnetic resonance data show that the L6RM results in significant chemical shift changes in loop 6 and surrounding regions, and that the binding of glycerol 3-phosphate (G3P) results in chemical shift changes for nuclei at the active site of the L6RM that are smaller than those of wild-type cTIM. Interactions with loop 6 of the L6RM stabilize the enediolate intermediate toward the elimination reaction catalyzed by the LDM. The LDM and L6RM result in 800000- and 23000-fold decreases, respectively, in kcat/Km for isomerization of GAP. Saturation of the LDM, but not the L6RM, by substrate and inhibitor phosphoglycolate is detected by steady-state kinetic analyses. We propose, on the basis of a comparison of X-ray crystal structures for wild-type TIM and the L6RM, that ligands bind weakly to the L6RM because a large fraction of the ligand binding energy is utilized to overcome destabilizing electrostatic interactions between the side chains of E168 and E129 that are predicted to develop in the loop-closed enzyme. Similar normalized yields of DHAP, d-DHAP, and d-GAP are formed in LDM- and L6RM-catalyzed reactions of GAP in D2O. The smaller normalized 12-13% yield of DHAP and d-DHAP observed for the mutant cTIM-catalyzed reactions compared with the 79% yield of these products for wild-type cTIM suggests that these mutations impair the transfer of a proton from O-2 to O-1 at the initial enediolate phosphate intermediate. No products are detected for the LDM-catalyzed isomerization reactions in D2O of [1-(13)C]GA and HPi, but the L6RM-catalyzed reaction in the presence of 0.020 M dianion gives a 2% yield of the isomerization product [2-(13)C,2-(2)H]GA.

Wildtype and engineered monomeric triosephosphate isomerase from Trypanosoma brucei: partitioning of reaction intermediates in D2O and activation by phosphite dianion.

Product yields for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D2O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this wildtype enzyme were determined by (1)H NMR spectroscopy and were compared with the yields determined in earlier work for the reactions catalyzed by TIM from rabbit and chicken muscle [O'Donoghue, A. C., Amyes, T. L., and Richard, J. P. (2005), Biochemistry 44, 2610 - 2621]. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-DHAP from isomerization with incorporation of deuterium from D2O into C-1 of DHAP, and d-GAP from incorporation of deuterium from D2O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate intermediate of the monoTIM reaction to form d-DHAP is less favorable ((k(C1))(D)/(k(C2))(D) = 1.1) than for the wildtype enzyme ((k(C1))(D)/(k(C2))(D) = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-(13)C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by (1)H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769-5778]. There is no detectable difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-(13)C]-GA in D2O. The kinetic parameters for phosphite dianion activation of the reactions of [1-(13)C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle [Amyes, T. L. and Richard, J. P. (2007) Biochemistry 46, 5841-5854], but there is no detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation of monoTIM back to the active structure for wildtype TIM.

Rescue of K12G triosephosphate isomerase by ammonium cations: the reaction of an enzyme in pieces.

The K12G mutation at yeast triosephosphate isomerase (TIM) results in a 5.5 × 10(5)-fold decrease in k(cat)/K(m) for isomerization of glyceraldehyde 3-phosphate, and the activity of this mutant can be successfully "rescued" by NH(4)(+) and primary alkylammonium cations. The transition state for the K12G mutant TIM-catalyzed reaction is stabilized by 1.5 kcal/mol by interaction with NH(4)(+). The larger 3.9 kcal/mol stabilization by CH(3)CH(2)CH(2)CH(2)NH(3)(+) is due to hydrophobic interactions between the mutant enzyme and the butyl side chain of the cation activator. There is no significant transfer of a proton from alkylammonium cations to GAP at the transition state for the K12G mutant TIM-catalyzed reaction, because activation by a series of RNH(3)(+) shows little or no dependence on the pK(a) of RNH(3)(+). A comparison of k(cat)/K(m) = 6.6 × 10(6) M(-1) s(-1) for the wildtype TIM-catalyzed isomerization of GAP and the third-order rate constant of 150 M(-2) s(-1) for activation by NH(4)(+) of the K12G mutant TIM-catalyzed isomerization shows that stabilization of the bound transition state by the effectively intramolecular interaction of the cationic side chain of Lys-12 at wildtype TIM is 6.3 kcal/mol greater than that for the corresponding intermolecular interaction of NH(4)(+) at K12G mutant TIM.

Bovine serum albumin-catalyzed deprotonation of [1-(13)C]glycolaldehyde: protein reactivity toward deprotonation of the alpha-hydroxy alpha-carbonyl carbon.

Bovine serum albumin (BSA) in D(2)O at 25 degrees C and pD 7.0 was found to catalyze the deuterium exchange reactions of [1-(13)C]glycolaldehyde ([1-(13)C]GA) to form [1-(13)C,2-(2)H]GA and [1-(13)C,2,2-di-(2)H]GA. The formation of [1-(13)C,2-(2)H]GA and [1-(13)C,2,2-di-(2)H]GA in a total yield of 51 +/- 3% was observed at early reaction times, and at later times, [1-(13)C,2-(2)H]GA was found to undergo BSA-catalyzed conversion to [1-(13)C,2,2-di-(2)H]GA. The overall second-order rate constant for these deuterium exchange reactions [(k(E))(P)] equals 0.25 M(-1) s(-1). By comparison, (k(E))(P) values of 0.04 M(-1) s(-1) [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769-5778] and 0.06 M(-1) s(-1) [Go, M. K., Koudelka, A., Amyes, T. L., and Richard, J. P. (2010) Biochemistry 49, 5377-5389] have been determined for the wild-type- and K12G mutant TIM-catalyzed deuterium exchange reactions of [1-(13)C]GA, respectively, to form [1-(13)C,2,2-di-(2)H]GA. These data show that TIM and BSA exhibit a modest catalytic activity toward deprotonation of the alpha-hydroxy alpha-carbonyl carbon. We suggest that this activity is intrinsic to many globular proteins, and that it must be enhanced to demonstrate meaningful de novo design of protein catalysts of proton transfer at alpha-carbonyl carbon.

Role of Lys-12 in catalysis by triosephosphate isomerase: a two-part substrate approach.

We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in (1) a approximately 50-fold increase in K(m) for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in K(i) for competitive inhibition by the intermediate analogue 2-phosphoglycolate, resulting from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and the ligand phosphodianion group; (2) a 12000-fold decrease in k(cat) for isomerization of GAP, suggesting a tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis complex to the transition state; and (3) a 6 x 10(5)-fold decrease in k(cat)/K(m), corresponding to a total 7.8 kcal/mol stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the K12G TIM-catalyzed isomerization of GAP in D(2)O were quantified as dihydroxyacetone phosphate (DHAP) (27%), [1(R)-(2)H]DHAP (23%), [2(R)-(2)H]GAP (31%), and methylglyoxal (18%) from an enzyme-catalyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of the transfer of a proton to the TIM-bound enediol(ate) intermediate in D(2)O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a >or=14-fold decrease in k(cat)/K(m) for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant enzyme-catalyzed reaction of [1-(13)C]GA in D(2)O is [1-(13)C,2,2-di-(2)H]GA from a nonspecific protein-catalyzed reaction. The observation that the K12G mutation results in a large decrease in k(cat)/K(m) for the reactions of both GAP and the neutral truncated substrate [1-(13)C]GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and the negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate "piece".

Hydron transfer catalyzed by triosephosphate isomerase. Products of the direct and phosphite-activated isomerization of [1-(13)C]-glycolaldehyde in D(2)O.

Product distributions for the reaction of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-(13)C]-GA) catalyzed by triosephosphate isomerase (TIM) in D(2)O at pD 7.0 in the presence of phosphite dianion and in its absence were determined by (1)H NMR spectroscopy. We observe three products for the relatively fast phosphite-activated reaction (Amyes, T. L., and Richard, J. P. (2007) Biochemistry 46, 5841-5854): [2-(13)C]-GA from isomerization with intramolecular transfer of hydrogen (12% of products), [2-(13)C,2-(2)H]-GA from isomerization with incorporation of deuterium from D(2)O at C-2 (64% of products), and [1-(13)C,2-(2)H]-GA from incorporation of deuterium from D(2)O at C-2 (23% of products). The much slower unactivated reaction in the absence of phosphite results in formation of the same three products along with the doubly deuterated product [1-(13)C,2,2-(2)H(2)]-GA. The two isomerization products ([2-(13)C]-GA and [2-(13)C,2-(2)H]-GA) are formed in the same relative yields in both the unactivated and the phosphite-activated reactions. However, the additional [1-(13)C,2-(2)H]-GA and the doubly deuterated [1-(13)C,2,2-(2)H(2)]-GA formed in the unactivated TIM-catalyzed reaction are proposed to result from nonspecific reaction(s) at the protein surface. The data provide evidence that phosphite dianion affects the rate, but not the product distribution, of the TIM-catalyzed reaction of [1-(13)C]-GA at the enzyme active site. They are consistent with the conclusion that both reactions occur at an unstable loop-closed form of TIM and that activation of the isomerization reaction by phosphite dianion results from utilization of the intrinsic binding energy of phosphite dianion to stabilize the active loop-closed enzyme.

Alanine-dependent reactions of 5'-deoxypyridoxal in water.

The non-enzymatic reaction of 5'-deoxypyridoxal (DPL) with l-alanine in water at 25 degrees C was investigated. DPL reacts with alanine to form an imine, which then undergoes deprotonation at the alpha-amino carbon of alanine to form a resonance delocalized DPL-stabilized carbanion. At early reaction times the only detectable products are pyruvate and the dimeric species formed by addition of the alpha-pyridine stabilized carbanion to DPL. No Claisen-type products of addition of the alpha-amino carbanion to DPL, as was previously reported to form from the reaction between DPL and glycine [K. Toth, T.L. Amyes, J.P. Richard, J.P.G. Malthouse, M.E. Ni Beilliu, J. Am. Chem. Soc. 126 (2004) 10538-10539], are observed. The electrophile reacts instead at the alpha-pyridyl carbon. This dimer is in chemical equilibrium with reactants. At longer reaction times about 50% of DPL is converted to 5'-deoxypyridoxamine, the thermodynamically favored product of formal transamination of DPL.