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Bacterial microcompartments are proteinaceous shells that encase specialized metabolic processes in bacteria. Recent advances in simplification of these intricate shells have encouraged bioengineering efforts. Here, we construct minimal shells derived from the Halothiobacillus neapolitanus α-carboxysome, which we term Cso-shell. Using cryogenic electron microscopy, the atomic-level structures of two shell forms were obtained, reinforcing notions of evolutionarily conserved features in bacterial microcompartment shell architecture. Encapsulation peptide sequences that facilitate loading of heterologous protein cargo within the shells were identified. We further provide a first demonstration in utilizing minimal bacterial microcompartment-derived shells for hosting heterologous enzymes. Cso-shells were found to stabilize enzymatic activities against heat shock, presence of methanol co-solvent, consecutive freeze-thawing, and alkaline environments. This study yields insights into α-carboxysome assembly and advances the utility of synthetic bacterial microcompartments as nanoreactors capable of stabilizing enzymes with varied properties and reaction chemistries.

In this work, a gold biosensor has been constructed and characterised in Escherichia coli ( E. coli ). Though gold biosensors have been previously reported, a NOT gate gold biosensing genetic circuit was constructed using an orthogonal inverter system as an alternative to a circuit where output is contingent solely on input. This enables the detection of changes in gold concentration whereby circuit remains OFF in presence of inducer and ON in absence of inducer. The proposed biosensor is an alternative to conventional forms of gold detection such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The constructed biosensor is a collection of components – golTSB operon (isolated from Salmonella enterica ), mf -Lon protease (isolated from Mesoplasma florum ) and associated ssrA degradation tag with GFP acting as a reporter. The minimum time and concentration required by the biosensor gol- mf lon-GFP (GMG) to achieve the ON output is characterised here using plate reader data derived from a single multi-stage assay.

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.

We have found enzymes from the berberine-bridge enzyme (BBE) superfamily (IPR012951) that catalyze the oxidative cyclization of the monoterpene moiety in cannabigerolic acid (CBGA) to form cannabielsoic acid B (CBSA). The enzymes are from a variety of organisms and are previously uncharacterized. This is the first report that describes enzymes that did not originate from the Cannabis plant that catalyze the production of cannabinoids. Out of 72 homologues chosen from the enzyme superfamily, six orthologues were shown to accept CBGA as a substrate and catalyze the biosynthesis of CBSA. The six enzymes discovered in this study are the first report of heterologous expression of BBEs that did not originate from the Cannabis plant that catalyze the production of cannabinoids using CBGA as substrate. This study details a new avenue for discovering and producing natural and unnatural cannabinoids.

Chromobacterium violaceum is a potential industrially relevant chassis due to its cyanogenic properties and diverse metabolic energetics. However, its application in synthetic biology is limited by the lack of characterised genetic parts. Six constitutive promoters of a 100-fold range in gene expression from the Anderson library were characterised in C. violaceum using an automated microplate reader. The datasheet provides an insight to the highly similar trend in promoter strengths in C. violaceum to the original characterised strengths of the Anderson library constitutive promoters in E. coli . This provides a foundation for constitutive promoters of different strengths as parts to be used for synthetic biology genetic constructs in C. violaceum .

In this work, we present a generalizable directed computational evolution protocol to effectively reduce the sequence space to be explored in rational enzyme design. The protocol involves in silico mutation modeling and substrate docking to rapidly identify mutagenesis hotspots that may enhance an enzyme's substrate binding and overall catalysis. By applying this protocol to a quorum-quenching Geobacillus kaustophilus lactonase, GKL, we generated 1,881 single mutants and docked high-energy intermediates of nine acyl homoserine lactones onto them. We found that Phe28 and Tyr99 were two hotspots that produced most of the predicted top 20 mutants. Of the 180 enzyme-substrate combinations (top 20 mutants × 9 substrates), 51 (28%) exhibited enhanced substrate binding and 22 (12%) had better overall activity when compared with wild-type GKL. X-ray crystallographic studies of Y99C and Y99P provided rationalized explanations for the enhancement in enzyme function and corroborated the utility of the protocol.

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.

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.

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 1H 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 ((kC1)D/(kC2)D = 1.1) than for the wildtype enzyme ((kC1)D/(kC2)D = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-13C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by 1H 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 [1-13C]-GA in D2O. The kinetic parameters for phosphite dianion activation of the reactions of [1-13C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle TIM [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, with the result that there is insufficient binding energy remaining to give a detectable stabilization of the transition state for the monoTIM-catalyzed reaction of [1-13C]-GA.

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.

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 aor=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".

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.

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

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.

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.

Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the “drug of last resort”. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.