Stephen L. Bearne

Inhibition of Escherichia coli glucosamine-6 phosphate synthase by reactive intermediate analogues - The role of the 2-amino function in catalysis

Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of d-glucosamine 6-phosphate fromd-fructose 6-phosphate using l-glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. The most potent carbohydrate-based inhibitor of GlmS reported to date is 2-amino-2-deoxy-d-glucitol 6-phosphate, an analogue of the putative cis-enolamine intermediate formed during catalysis. The interaction of a series of structurally related cis-enolamine intermediate analogues with GlmS is described. Although arabinose oxime 5-phosphate is identified as a good competitive inhibitor of GlmS with an inhibition constant equal to 1.2 (±0.3) mm, the presence of the amino function at the 2-position is shown to be important for potent inhibition. Comparison of the binding affinities of 2-deoxy-d-glucitol 6-phosphate and 2-amino-2-deoxy-d-glucitol 6-phosphate indicates that the amino function contributes −4.1 (±0.1) kcal/mol to the free energy of inhibitor binding. Similarly, comparison of the binding affinities of 2-deoxy-d-glucose 6-phosphate andd-glucosamine 6-phosphate indicates that the amino function contributes −3.0 (±0.1) kcal/mol to the free energy of product binding. Interactions between GlmS and the 2-amino function of its ligands contribute to the uniform binding of the product and thecis-enolamine intermediate as evidenced by the similar contribution of the amino group to the free energy of binding ofd-glucosamine 6-phosphate and 2-amino-2-deoxy-d-glucitol 6-phosphate, respectively.

Proteomic investigation of amino acid catabolism in the indigenous gut anaerobe Fusobacterium varium

The butyrate‐producing anaerobe Fusobacterium varium is an integral constituent of human gut microflora. Unlike many gut microorganisms, F. varium is capable of fermenting both amino acids and glucose. Although F. varium has been implicated in beneficial as well as pathological bacterium–host interactions, its genome has not been sequenced. To obtain a better understanding of the metabolic processes associated with amino acid fermentation by F. varium, we used a gel‐based proteomic approach to examine the changes in the soluble proteome accompanying the utilization of eight different growth substrates: glucose, L‐ and D‐glutamate, L‐histidine, L‐ and D‐lysine, and L‐ and D‐serine. Using LC‐MS/MS to analyze ˜25% of the detected protein spots, we were able to identify 47 distinct proteins. While the intracellular concentrations of enzymes characteristic of a catabolic pathway for a specific amino acid were selectively increased in response to the presence of an excess of that amino acid in the growth medium, the concentrations of the core acetate–butyrate pathway enzymes remained relatively constant. Our analysis revealed (i) high intracellular concentrations of glutamate mutase and β‐methylaspartate ammonia‐lyase under all growth conditions, underscoring the importance of the methylaspartate pathway of glutamate catabolism in F. varium (ii) the presence of two enzymes of the hydroxyglutarate pathway of glutamate degradation in the proteome of F. varium ((R)‐2‐hydroxyglutaryl‐CoA dehydratase and NAD‐specific glutamate dehydrogenase) specifically when L‐glutamate was the main energy source (iii) the presence of genes in the genome of F. varium encoding each of the enzymes of the hydroxyglutarate pathway (iv) the presence of both L‐ and D‐serine ammonia‐lyases (dehydratases) which permit F. varium to thrive on either L‐ or D‐serine, respectively, and (v) the presence of aspartate‐semialdehyde dehydrogenase and dihydrodipicolinate synthase, consistent with the ability of F. varium to synthesize meso‐2,6‐diaminopimelic acid as a component of its peptidoglycan. Proteins involved in other cellular processes, including oxidation–reduction reactions, protein synthesis and turnover, and transport were also identified.

Oxidative stress response in the opportunistic oral pathogen Fusobacterium nucleatum

The anaerobic, Gram‐negative bacillus Fusobacterium nucleatum plays a vital role in oral biofilm formation and the development of periodontal disease. The organism plays a central bridging role between early and late colonizers within dental plaque and plays a protective role against reactive oxygen species. Using a two‐dimensional gel electrophoresis and mass spectrometry approach, we have annotated 78 proteins within the proteome of F. nucleatum subsp. nucleatum and identified those proteins whose apparent intracellular concentrations change in response to either O2‐ or H2O2‐induced oxidative stress. Three major protein systems were altered in response to oxidative stress: (i) proteins of the alkyl hydroperoxide reductase/thioredoxin reductase system were increased in intracellular concentration; (ii) glycolytic enzymes were modified by oxidation (i.e. D‐glyceraldehyde 3‐phosphate dehydrogenase, and fructose 6‐phosphate aldolase) or increased in intracellular concentration, with an accompanying decrease in ATP production; and (iii) the intracellular concentrations of molecular chaperone proteins and related proteins (i.e. ClpB, DnaK, HtpG, and HrcA) were increased.

Aspartate-107 and leucine-109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by Escherichia coli CTP synthase

CTP synthase catalyses the ATP-dependent formation of CTP from UTP using either NH(3) or L-glutamine as the nitrogen source. GTP is required as an allosteric effector to promote glutamine hydrolysis. In an attempt to identify nucleotide-binding sites, scanning alanine mutagenesis was conducted on a highly conserved region of amino acid sequence (residues 102-118) within the synthase domain of Escherichia coli CTP synthase. Mutant K102A CTP synthase exhibited wild-type activity with respect to NH(3) and glutamine; however, the R105A, D107A, L109A and G110A enzymes exhibited wild-type NH(3)-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation. The E103A, R104A and H118A enzymes exhibited no glutamine-dependent activity and were only partially active with NH(3). Although these observations were compatible with impaired activation by GTP, the apparent affinity of the D107A, L109A and G110A enzymes for GTP was reduced only 2-4-fold, suggesting that these residues do not play a significant role in GTP binding. In the presence of GTP, the k (cat) values for glutamine hydrolysis by the D107A and L109A enzymes were identical with that of wild-type CTP synthase. Overall, the kinetic properties of L109A CTP synthase were consistent with an uncoupling of glutamine hydrolysis from CTP formation that occurs because an NH(3) tunnel has its normal structure altered or fails to form. L109F CTP synthase was prepared to block totally the putative NH(3) tunnel; however, this enzymes rate of glutamine-dependent CTP formation and glutaminase activity were both impaired. In addition, we observed that mutation of amino acids located between residues 102 and 118 in the synthase domain can affect the enzymes glutaminase activity, suggesting that these residues interact with residues in the glutamine amide transfer domain because they are in close proximity or via a conformationally dependent signalling mechanism.

Structure of mandelate racemase with bound intermediate analogues benzohydroxamate and cupferron.

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, to 2.2-Å resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state and/or intermediate because its binding affinity for 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and Cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, Cupferron, and the ground state analogue (S)-atrolactate reveal that the para carbon of the substrate phenyl ring moves by 0.8-1.2 Å between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to that of MR with bound (S)-atrolactate, the intermediate-Mg(2+) distance becomes shorter, suggesting a tighter complex with the catalytic Mg(2+). In addition, Tyr 54 moves closer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle.

Purification of recombinant mandelate racemase: Improved catalytic activity

Mandelate racemase (MR, E.C. 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate and has been studied extensively as a model for understanding how enzymes catalyze the deprotonation of carbon acid substrates with relatively high pK(a) values. Purification of recombinant MR as a fusion protein with an N-terminal hexahistidine tag using immobilized-nickel ion affinity chromatography and elution with a linear gradient of EDTA revealed three enzyme species (mrI, mrII, and mrIII). While mrIII was catalytically inactive, both mrI and mrII catalyzed the racemization of (S)-mandelate with turnover numbers (k(cat)) of 190+/-22 and 940+/-24s(-1), respectively. Circular dichroism analysis suggested that mrIII was a partially unfolded or misfolded form of the enzyme. Replacement of the N-terminal hexahistidine tag by a StrepII-tag appeared to ameliorate the folding problem yielding a single enzyme species with a turnover number of 1124+/-43s(-1). The MR fusion protein bearing an N-terminal StrepII-tag and a C-terminal decahistidine tag also exhibited reduced turnover (k(cat)=472+/-37s(-1)). These results highlight a potential problem that may be encountered when producing fusion enzymes bearing a polyhistidine tag: soluble, active enzyme may be obtained but care must be taken to ensure that it is free of minor misfolded forms that can alter the apparent activity of the enzyme.

Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax

Cytidine 5′-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH3 or l-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mm and NH3-dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP ≈ 6-thio-GTP > ITP ≈ guanosine 5′-tetraphosphate > O6-methyl-GTP > 2′-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2′,3′-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5′-triphosphate, 2′-OH, and 3′-OH are required for full activation. The 2-NH2 group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH2 group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH3- and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC50 ≈ 0.2-0.5 mm). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC50 ≈ 4-12 mm). 8-Oxoguanosine was the best inhibitor identified (IC50 = 80 μm). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5′-triphosphate synthase inhibitors.

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

alpha-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279nm in the presence of octyl-beta-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K(m)=48+/-5microM, k(cat)=291+/-30s(-1)), but turned over (2S)-ibuprofenoyl-CoA (K(m)=86+/-6microM, k(cat)=450+/-14s(-1)) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His(6)-tag had a catalytic efficiency (k(cat)/K(m)) that was reduced 22% and 47% in the 2S-->2R and 2R-->2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.

Potent Inhibition of Mandelate Racemase by a Fluorinated Substrate-Product Analogue with a Novel Binding Mode

Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Because trifluorolactate is also a substrate of MR, we anticipated that replacing the phenyl rings of the competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl groups might furnish an inhibitor. Surprisingly, the substrate-product analogue 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoate (TFHTP) was a potent competitive inhibitor [Ki = 27 ± 4 μM; cf. Km = 1.2 mM for both (R)-mandelate and (R)-trifluorolactate]. To understand the origins of this high binding affinity, we determined the X-ray crystal structure of the MR-TFHTP complex to 1.68 Å resolution. Rather than chelating the active site Mg(2+) with its glycolate moiety, like other ground state analogues, TFHTP exhibited a novel binding mode with the two trifluoromethyl groups closely packed against the 20s loop and the carboxylate bridging the two active site Brønsted acid-base catalysts Lys 166 and His 297. Recognizing that positioning a carboxylate between the Brønsted acid-base catalysts could yield an inhibitor, we showed that tartronate was a competitive inhibitor of MR (Ki = 1.8 ± 0.1 mM). The X-ray crystal structure of the MR-tartronate complex (1.80 Å resolution) revealed that the glycolate moiety of tartronate chelated the Mg(2+) and that the carboxylate bridged Lys 166 and His 297. Models of tartronate in monomers A and B of the crystal structure mimicked the binding orientations of (S)-mandelate and that anticipated for (R)-mandelate, respectively. For the latter monomer, the 20s loop appeared to be disordered, as it also did in the X-ray structure of the MR triple mutant (C92S/C264S/K166C) complexed with benzilate, which was determined to 1.89 Å resolution. These observations indicate that the 20s loop likely undergoes a significant conformational change upon binding (R)-mandelate. In general, our observations suggest that inhibitors of other enolase superfamily enzymes may be designed to capitalize on the recognition of the active site Brønsted acid-base catalysts as binding determinants.

Inhibition of glutamate racemase by substrate-product analogues.

D-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of D-glutamate from L-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate-product analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part, for periodontal disease and colorectal cancer (Ki=3.1±0.6 mM, cf. Km=1.41±0.06 mM). The cyclic substrate-product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor, giving only ∼30% inhibition at a concentration of 40 mM. The related cyclic substrate-product analogue 1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (Ki=18.4±1.2 mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimicking the structure of both enantiomeric substrates (substrate-product analogues) serves as a useful design strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve as potential lead compounds for further development.