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The ydj gene cluster is annotated to catalyze the catabolism of an unknown carbohydrate. Previously, YdjI, a class II aldolase, was shown to catalyze the retro-aldol cleavage of l- glycero -l- galacto -octuluronate-1-phosphate into DHAP and l-arabinuronate. In this report, the functional characterization of YdjH is presented. YdjH catalyzes the phosphorylation of 2-keto-monosaccharides at the C1 hydroxyl group with a substrate profile significantly more stringent than that of YdjI. Similar to YdjI, YdjH shows a strong preference for higher-order monosaccharides (seven to nine carbons) with a carboxylate terminus. The best substrate was determined to be l- glycero -l- galacto -octuluronate, yielding l- glycero -l- galacto -octuluronate-1-phosphate with a k cat of 16 s -1 and a k cat / K m of 2.1 × 10 4 M -1 s -1 . This is apparently the first reported example of kinase activity with eight-carbon monosaccharides. Two crystal structures of YdjH were previously determined to 2.15 and 1.8 Å resolution (Protein Data Bank entries 3H49 and 3IN1 ). We present an analysis of the active site layout and use computational docking to identify potential key residues in the binding of l- glycero -l- galacto -octuluronate.

The ydj gene cluster is found in 80% of sequenced Escherichia coli genomes and other closely related species in the human microbiome. On the basis of the annotations of the enzymes located in this cluster, it is expected that together they catalyze the catabolism of an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the ydj gene cluster of E. coli K-12. It is predicted to be a class II aldolase of unknown function. Here we describe a structural and functional characterization of this enzyme. YdjI catalyzes the hydrogen/deuterium exchange of the pro- S hydrogen at C3 of dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol condensation with a variety of aldo sugars. YdjI shows a strong preference for higher-order (seven-, eight-, and nine-carbon) monosaccharides with specific hydroxyl stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best substrate is l-arabinuronic acid with an apparent k cat of 3.0 s -1 . The product, l- glycero -l- galacto -octuluronate-1-phosphate, has a k cat / K m value of 2.1 × 10 3 M -1 s -1 in the retro-aldol reaction with YdjI. This is the first recorded synthesis of l- glycero -l- galacto -octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI, determined to a nominal resolution of 1.75 Å (Protein Data Bank entry 6OFU ), reveals unusual positions for two arginine residues located near the active site. Computational docking was utilized to distinguish preferable binding orientations for l- glycero -l- galacto -octuluronate-1-phosphate. These results indicate a possible alternative binding orientation for l- glycero -l- galacto -octuluronate-1-phosphate compared to that observed in other class II aldolases, which utilize shorter carbohydrate molecules.

Lytic transglycosylases (LTs) are bacterial enzymes that catalyze the cleavage of the glycan strands of the bacterial cell wall. The mechanism of this cleavage is a remarkable intramolecular transacetalization reaction, accomplished by an ensemble of active-site residues. Because the LT reaction occurs in parallel with the cell wall bond-forming reactions catalyzed by the penicillin-binding proteins, simultaneous inhibition of both enzymes can be particularly bactericidal to Gram-negative bacteria. The MltE lytic transglycosylase is the smallest of the eight LTs encoded by the Escherichia coli genome. Prior crystallographic and computational studies identified four active-site residues-E64, S73, S75, and Y192-as playing roles in catalysis. Each of these four residues was individually altered by mutation to give four variant enzymes (E64Q, S73A, S75A, and Y192F). All four variants showed reduced catalytic activity [soluble wild type (100%) > soluble Y192F and S75A (both 40%) > S73A (4%) > E64Q (≤1%)]. The crystal structure of each variant protein was determined at the resolution of 2.12 Å for E64Q, 2.33 Å for Y192F, 1.38 Å for S73A, and 1.35 Å for S75A. These variants show alteration of the hydrogen-bond interactions of the active site. Within the framework of a prior computational study of the LT mechanism, we suggest the mechanistic role of these four active-site residues in MltE catalysis.

The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes ( ycjM-W and ompG) of unknown function in Escherichia coli K-12 were determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (α-(1,2)-d-glucose-d-glucose), this enzyme catalyzes the formation of d-glucose and β-d-glucose-1-phosphate ( k cat = 1.1 s -1 , K m = 1.05 mM, and k cat / K m = 1.12 × 10 3 M -1 s -1 ). Additionally, it was also shown that in the presence of β-d-glucose-1-phosphate, YcjT can catalyze the formation of other disaccharides using 1,5-anhydro-d-glucitol, l-sorbose, d-sorbitol, or l-iditol as a substitute for d-glucose. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a β-phosphoglucomutase that catalyzes the isomerization of β-d-glucose-1-phosphate ( k cat = 21 s -1 , K m = 18 μM, and k cat / K m = 1.1 × 10 6 M -1 s -1 ) to d-glucose-6-phosphate. YcjU was also shown to exhibit catalytic activity with β-d-allose-1-phosphate, β-d-mannose-1-phosphate, and β-d-galactose-1-phosphate. YcjM catalyzes the phosphorolysis of α-(1,2)-d-glucose-d-glycerate with a k cat = 2.1 s -1 , K m = 69 μM, and k cat / K m = 3.1 × 10 4 M -1 s -1 .

Recent advances in proteomics and genomics have enabled discovery of thousands of previously nonannotated small open reading frames (smORFs) in genomes across evolutionary space. Furthermore, quantitative mass spectrometry has recently been applied to analysis of regulated smORF expression. However, bottom-up proteomics has remained relatively insensitive to membrane proteins, suggesting they may have been underdetected in previous studies. In this report, we add biochemical membrane protein enrichment to our previously developed label-free quantitative proteomics protocol, revealing a never-before-identified heat shock protein in Escherichia coli K12. This putative smORF-encoded heat shock protein, GndA, is likely to be ∼36-55 amino acids in length and contains a predicted transmembrane helix. We validate heat shock-regulated expression of the gndA smORF and demonstrate that a GndA-GFP fusion protein cofractionates with the cell membrane. Quantitative membrane proteomics therefore has the ability to reveal nonannotated small proteins that may play roles in bacterial stress responses.

l-Aspartate oxidase, encoded by the nadB gene, is the first enzyme in the de novo synthesis of NAD + in bacteria. This FAD-dependent enzyme catalyzes the oxidation of l-aspartate to generate iminoaspartate and reduced flavin. Distinct from most amino acid oxidases, it can use either molecular oxygen or fumarate to reoxidize the reduced enzyme. Sequence alignments and the three-dimensional crystal structure have revealed that the overall fold and catalytic residues of NadB closely resemble those of the succinate dehydrogenase/fumarate reductase family rather than those of the prototypical d-amino acid oxidases. This suggests that the enzyme can catalyze amino acid oxidation via typical amino acid oxidase chemistry, involving the removal of protons from the α-amino group and the transfer of the hydride from C2, or potentially deprotonation at C3 followed by transfer of the hydride from C2, similar to chemistry occurring during succinate oxidation. We have investigated this potential mechanistic ambiguity using a combination of primary, solvent, and multiple deuterium kinetic isotope effects in steady state experiments. Our results indicate that the chemistry is similar to that of typical amino acid oxidases in which the transfer of the hydride from C2 of l-aspartate to FAD is rate-limiting and occurs in a concerted manner with respect to deprotonation of the α-amine. Together with previous kinetic and structural data, we propose that NadB has structurally evolved from succinate dehydrogenase/fumarate reductase-type enzymes to gain the new functionality of oxidizing amino acids while retaining the ability to reduce fumarate.

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

TrpH or YciV (locus tag b1266) from Escherichia coli is annotated as a protein of unknown function that belongs to the polymerase and histidinol phosphatase (PHP) family of proteins in the UniProt and NCBI databases. Enzymes from the PHP family have been shown to hydrolyze organophosphoesters using divalent metal ion cofactors at the active site. We found that TrpH is capable of hydrolyzing the 3'-phosphate from 3',5'-bis-phosphonucleotides. The enzyme will also sequentially hydrolyze 5'-phosphomononucleotides from 5'-phosphorylated RNA and DNA oligonucleotides, with no specificity toward the identity of the nucleotide base. The enzyme will not hydrolyze RNA or DNA oligonucleotides that are unphosphorylated at the 5'-end of the substrate, but it makes no difference whether the 3'-end of the oligonucleotide is phosphorylated. These results are consistent with the sequential hydrolysis of 5'-phosphorylated mononucleotides from oligonucleotides in the 5' → 3' direction. The catalytic efficiencies for hydrolysis of 3',5'-pAp, p(Ap)A, p(Ap)4A, and p(dAp)4dA were determined to be 1.8 × 10(5), 9.0 × 10(4), 4.6 × 10(4), and 2.9 × 10(3) M(-1) s(-1), respectively. TrpH was found to be more efficient at hydrolyzing RNA oligonucleotides than DNA oligonucleotides. This enzyme can also hydrolyze annealed DNA duplexes, albeit at a catalytic efficiency approximately 10-fold lower than that of the corresponding single-stranded oligonucleotides. TrpH is the first enzyme from E. coli that has been found to possess 5' → 3' exoribonuclease activity. We propose to name this enzyme RNase AM.

There are five distinct core structures in the lipopolysaccharides of Escherichia coli and at least two in Salmonella isolates, which vary principally in the outer core oligosaccharide. Six outer core glycosyltransferases, E. coli K-12 WaaG, WaaB, and WaaO and Salmonella typhimurium WaaI, WaaJ, and WaaK, were cloned, overexpressed, and purified. A novel substrate for WaaG was isolated from ΔwaaG E. coli overexpressing the lipid A phosphatase lpxE and the lipid A late acyltransferase lpxM. The action of lpxE and lpxM in the ΔwaaG background yielded heptose2-1-dephospho Kdo2-lipid A, a 1-dephosphorylated hexa-acylated lipid A with the inner core sugars that is easily isolated by organic extraction. Using this structurally defined acceptor and commercially available sugar nucleotides, each outer core glycosyltransferases was assayed in vitro. We show that WaaG and WaaB add a glucose and galactose sequentially to heptose2-1-dephospho Kdo2-lipid A. E. coli K-12 WaaO and S. typhimurium WaaI add a galactose to the WaaG/WaaB product but can also add a galactose to the WaaG product directly without the branched core sugar added by WaaB. Both WaaI and WaaO require divalent metal ions for optimal activity; however, WaaO, unlike WaaI, can add several glucose residues to its lipid acceptor. Using the product of WaaG, WaaB, and WaaI, we show that S. typhimurium WaaJ and WaaK transfer a glucose and N-acetylglucosamine, respectively, to yield the full outer core. This is the first demonstration of the in vitro assembly of the outer core of the lipopolysaccharide using defined lipid A-oligosaccharide acceptors and sugar donors.

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps.

Siderophore-interacting proteins (SIPs), such as YqjH from Escherichia coli, are widespread among bacteria and commonly associated with iron-dependent induction and siderophore utilization. In this study, we show by detailed biochemical and genetic analyses the reaction mechanism by which the YqjH protein is able to catalyze the release of iron from a variety of iron chelators, including ferric triscatecholates and ferric dicitrate, displaying the highest efficiency for the hydrolyzed ferric enterobactin complex ferric (2,3-dihydroxybenzoylserine)(3). Site-directed mutagenesis revealed that residues K55 and R130 of YqjH are crucial for both substrate binding and reductase activity. The NADPH-dependent iron reduction was found to proceed via single-electron transfer in a double-displacement-type reaction through formation of a transient flavosemiquinone. The capacity to reduce substrates with extremely negative redox potentials, though at low catalytic rates, was studied by displacing the native FAD cofactor with 5-deaza-5-carba-FAD, which is restricted to a two-electron transfer. In the presence of the reconstituted noncatalytic protein, the ferric enterobactin midpoint potential increased remarkably and partially overlapped with the effective E(1) redox range. Concurrently, the observed molar ratios of generated Fe(II) versus NADPH were found to be ~1.5-fold higher for hydrolyzed ferric triscatecholates and ferric dicitrate than for ferric enterobactin. Further, combination of a chromosomal yqjH deletion with entC single- and entC fes double-deletion backgrounds showed the impact of yqjH on growth during supplementation with ferric siderophore substrates. Thus, YqjH enhances siderophore utilization in different iron acquisition pathways, including assimilation of low-potential ferric substrates that are not reduced by common cellular cofactors.

The dapdiamides make up a family of antibiotics that have been presumed to be cleaved in the target cell to enzyme-inhibitory N-acyl-2,3-diaminopropionate (DAP) warheads containing two alternative electrophilic moieties. Our prior biosynthetic studies revealed that an eneamide warhead is made first and converted to an epoxyamide via a three-enzyme branch pathway. Here we provide a rationale for this logic. We report that the R,R-epoxyamide warhead is a more efficient covalent inactivator of glucosamine-6-phosphate synthase by 1 order of magnitude versus the eneamide, and this difference correlates with a >10-fold difference in antibiotic activity for the corresponding acyl-DAP dipeptides.

Two different scenarios for the recruitment of evolutionary starting points and their subsequent divergence to give new enzymes have been described. The coincidental, promiscuous starting activity may regard the same reaction chemistry on a new substrate (substrate ambiguity). Alternatively, substrate binding guides the recruitment of an enzyme whose reaction chemistry differs from that of the newly evolving one (catalytic promiscuity). While substrate ambiguity seems to underlie the divergence of most enzyme families, the relative levels of occurrence of these scenarios remain unknown. Screening the Escherichia coli proteome with a comparative series of xenobiotic substrates, we found that substrate ambiguity was, as anticipated, more frequent than reaction promiscuity. However, for at least one unnatural reaction (phosphonoesterase), a promiscuous enzyme was identified only when the substrate was decorated with the naturally abundant phosphate group. These findings support the prevailing hypothesis of chemistry-driven divergence but also suggest that recognition of familiar substrate motifs plays a role. In the absence of enzymes catalyzing the same chemistry, having a familiar, naturally occurring substrate motif (chemophore) such as phosphate may increase the likelihood of catalytic promiscuity. Chemophore anchoring may also find practical applications in identifying catalysts for unnatural reactions.

The Escherichia coli siderophore enterobactin is synthesized in response to iron starvation. 2,3-Dihydro-2,3-dihydroxybenzoate dehydrogenase (EntA) produces 2,3-dihydroxybenzoate (DHB), a biosynthetic intermediate. 2,3-Dihydroxybenzoate-AMP ligase (EntE) adenylates DHB, activating it for attachment to the NRPS substrate holo-EntB. Using analytical ultracentrifugation, we found that EntA undergoes concentration-dependent dimer-tetramer self-association (K(D) = 12.3 μM). We further found that EntA can form a specific complex with EntE. Pull-down assays revealed that recombinant EntA bait pulled down EntE from E. coli lysates, whereas recombinant EntE bait could pull down EntA. Addition of the SMCC cross-linker to a mixture of EntA and EntE resulted in a cross-linked product with a molecular mass of >250 kDa, suggesting a complex stoichiometry of one EntA tetramer and four EntE monomers. The effect of EntA on EntE activity was also examined. Addition of a 4-fold excess of EntA to an EntE assay mixture resulted in a 6-fold stimulation of EntE activity. EntA was also found to perturb the FRET signal between EntE donor residues and EntE-bound DHB. By following the EntA-dependent decrease in the magnitude of the EntE-DHB FRET signal, EntA-EntE binding behavior was found to be sigmoidal, suggesting the presence of both low- and high-affinity binding sites. The EntA-EntE interaction was also directly measured by isothermal titration calorimetry at 10 °C. The resulting binding isotherm fit well to a model describing two binding sites, supporting our AUC and fluorescence data. Taken together, our data show that tetrameric EntA optimally interacts with EntE, resulting in an enhancement of EntE activity.

Thioredoxin-1 from Escherichia coli has frequently been used as a model substrate in protein folding studies. However, for reasons of convenience, these studies have focused largely on oxidized thioredoxin and not on reduced thioredoxin, the more physiologically relevant species. Here we describe the first extensive characterization of the refolding kinetics and conformational thermodynamics of reduced thioredoxin. We have previously described a genetic screen that yielded mutant thioredoxin proteins that fold more slowly in both the oxidized and reduced forms. In this study, we apply our more detailed analysis of reduced thioredoxin folding to a larger number of folding mutants that includes those obtained from continuation of the genetic screen. We have identified mutant proteins that display folding defects specifically in the reduced state but not the oxidized state. Some of these substitutions represent unusual folding mutants in that they result in semiconservative substitutions at solvent-exposed positions in the folded conformation and do not appear to affect the conformational stability of the protein. Further, the genetic selection yields mutants at only a limited number of sites, pointing to perhaps the most critical amino acids in the folding pathway and underscoring, in particular, the role of the carboxy-terminal amino acids in the folding of thioredoxin. Our results demonstrate the importance of studying the physiologically relevant folding species.

Escherichia coli mutants deficient in 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) biosynthesis are conditionally lethal, but their phenotypes are bypassed by certain suppressor mutations or by overexpression of MsbA, the inner membrane flippase for core-lipid A. These strains grow on broth with the tetraacylated precursor lipid IV(A) replacing lipopolysaccharide [Meredith, T. C., et al. (2006) ACS Chem. Biol. 1, 33-42]. Deletion of kdtA, which encodes the Kdo transferase, is possible under these conditions. We now show that lipid IV(A) reaches the outer surface of the outer membrane in these strains, as judged by its accessibility to the lipase PagL. On the assumption that MsbA is optimized to transport penta- or hexaacylated lipid A, we overexpressed the lauroyl- or the myristoyltransferase of lipid A biosynthesis, encoded by lpxL and lpxM, respectively, and demonstrated that kdtA deletion mutants were also viable in this setting. Although E. coli LpxL is stimulated by the presence of the Kdo disaccharide in its acceptor substrate, LpxL does slowly acylate lipid IV(A). Overexpression of LpxL from a plasmid suppressed the lethality of kdtA deletions on nutrient broth at 30 or 37 degrees C without the need for MsbA overproduction. These strains accumulated penta- and hexaacylated free lipid A containing a secondary laurate chain or a laurate and a myristate chain, respectively. Deletion of kdtA in strains overexpressing LpxM accumulated pentaacylated lipid A with a secondary myristate moiety. None of the strains lacking kdtA grew in the presence of bile salts at any temperature or on nutrient broth at 42 degrees C. Our findings show that the main function of Kdo is to provide the right substrates for the acyltransferases LpxL and LpxM, resulting in the synthesis of penta- and hexaacylated lipid A, which is optimal for the MsbA flippase.

ATP hydrolysis by the maltose transporter (MalFGK(2)) is regulated by maltose binding protein (MBP). Binding of maltose to MBP brings about a conformational change from open to closed that leads to a strong stimulation of the MalFGK(2) ATPase. In this study, we address the long-standing but enigmatic observation that unliganded MBP is also able to stimulate MalFGK(2). Although the mechanism of this stimulation is not understood, it is sometimes attributed to a small amount of closed (but unliganded) MBP that may exist in solution. To gain insight into how MBP regulates the MalFGK(2) ATPase, we have investigated whether the open or the closed conformation of MBP is responsible for MalFGK(2) stimulation in the absence of maltose. The effect of MBP concentration on the stimulation of MalFGK(2) was assessed: for unliganded MBP, the apparent K(M) for stimulation of MalFGK(2) was below 1 microM, while for maltose-bound MBP, the K(M) was approximately 15 microM. We show that engineered MBP molecules in which the open-closed equilibrium has been shifted toward the closed conformation have a decreased ability to stimulate MalFGK(2). These results indicate that stimulation of the MalFGK(2) ATPase by unliganded MBP does not proceed through a closed conformation and instead must operate through a different mechanism than stimulation by liganded MBP. One possible explanation is that the open conformation is able to activate the MalFGK(2) ATPase directly.

One of the major challenges in the postgenomic era is the functional assignment of proteins using sequence- and structure-based predictive methods coupled with experimental validation. We have used these approaches to investigate the structure and function of the Escherichia coli K-12 protein YfaU, annotated as a putative 4-hydroxy-2-ketoheptane-1,7-dioate aldolase (HpcH) in the sequence databases. HpcH is the final enzyme in the degradation pathway of the aromatic compound homoprotocatechuate. We have determined the crystal structure of apo-YfaU and the Mg (2+)-pyruvate product complex. Despite greater sequence and structural similarity to HpcH, genomic context suggests YfaU is instead a 2-keto-3-deoxy sugar aldolase like the homologous 2-dehydro-3-deoxygalactarate aldolase (DDGA). Enzyme kinetic measurements show activity with the probable physiological substrate 2-keto-3-deoxy- l-rhamnonate, supporting the functional assignment, as well as the structurally similar 2-keto-3-deoxy- l-mannonate and 2-keto-3-deoxy- l-lyxonate (see accompanying paper: Rakus, J. F., Fedorov, A. A., Fedorov, E. V., Glasner, M. E., Hubbard, B. K., Delli, J. D., Babbitt, P. C., Almo, S. C., and Gerlt, J. A. (2008) Biochemistry 47, 9944-9954). YfaU has similar activity toward the HpcH substrate 4-hydroxy-2-ketoheptane-1,7-dioate and synthetic substrates 4-hydroxy-2-ketopentanoic acid and 4-hydroxy-2-ketohexanoic acid. This indicates a relaxed substrate specificity that complicates the functional assignment of members of this enzyme superfamily. Crystal structures suggest these enzymes use an Asp-His intersubunit dyad to activate a metal-bound water or hydroxide for proton transfer during catalysis.

Enzymes that share the (beta/alpha) 8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal (beta/alpha) 2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth beta-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, DeltaT196, DeltaS197 and DeltaG198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in k cat/ K m are dominated by changes in k cat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate group hydrogen bonds not only with the conserved motif but also with an active site loop following the sixth beta-strand, providing a potential structural mechanism for coupling substrate binding with catalysis.

The d-allose and N-acetyl-d-mannosamine kinases of Escherichia coli K-12 are divergent members of the functionally diverse ROK (repressor, open reading frame, kinase) superfamily. Previous work in our laboratory has demonstrated that AlsK and NanK possess weak phosphoryl transfer activity toward the alternate substrate d-glucose. To gain insight into the evolutionary mechanisms that fuel the specialization of individual enzyme function, experimental laboratory evolution was conducted to improve the glucokinase activities of AlsK and NanK. Error-prone PCR was combined with in vivo functional selection in a glucokinase-deficient bacterium to identify four independent single nucleotide substitutions in the alsK and nanK genes that improve the glucokinase activity of each enzyme. The most advantageous substitutions, L84P in NanK and A73G in AlsK, enhance the kcat/Km values for phosphoryl transfer to glucose by 12-fold and 60-fold, respectively. Both substitutions co-localize to a variable loop region located between the fourth beta-sheet and the second alpha-helix of the ROK scaffold. A multiple sequence alignment of diverse ROK family members reveals that the A73G substitution in AlsK recapitulates a conserved glycine residue present in many ROK proteins, including some transcriptional repressors. Steady-state kinetic analyses of the selected AlsK and NanK variants demonstrate that their native activities toward d-allose and N-acetyl-d-mannosamine are largely unaffected by the glucokinase-enhancing substitutions. Substrate specificity profiling reveals that the A73G AlsK and L84P NanK variants display systematic improvements in the kcat/Km values for a variety of nonnative carbohydrates. This finding is consistent with an evolutionary process that includes the formation of intermediates possessing relaxed substrate specificities during the initial steps of enzyme functional divergence.

The genes encoding metabolic enzymes involved in glucose metabolism, the TCA cycle, and biosynthesis of amino acids, purines, pyrimidines, and cofactors would be expected to be essential for growth of Escherichia coli on glucose because the cells must synthesize all of the building blocks for cellular macromolecules. Surprisingly, 80 of 227 of these genes are not essential. Analysis of why these genes are not essential provides insights into the metabolic sophistication of E. coli and into the evolutionary pressures that have shaped its physiology. Alternative routes enabled by interconnecting pathways can allow a defective step to be bypassed. Isozymes, alternative enzymes, broad-specificity enzymes, and multifunctional enzymes can often substitute for a missing enzyme. We expect that the apparent redundancy in these metabolic pathways has arisen due to the need for E. coli to survive in a variety of habitats and therefore to have a metabolism that allows optimal exploitation of varying environmental resources and synthesis of small molecules when they cannot be obtained from the environment.

ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the interconversion of ADP-beta-d-glycero-d-manno-heptose (ADP-d,d-Hep) and ADP-beta-l-glycero-d-manno-heptose (ADP-l,d-Hep). The latter compound provides the heptose moiety that is used in lipopolysaccharide biosynthesis by Gram-negative bacteria. Several lines of evidence suggest that the enzyme uses a direct oxidation/reduction mechanism involving a tightly bound NADP+ cofactor. An initial oxidation at C-6'' gives a 6''-keto intermediate, and a subsequent reduction on the opposite face of the carbonyl group generates the epimeric product. The reorientation required for the nonstereoselective reduction could take place within a single active site, or it could involve the release of the intermediate and rebinding in an altered conformation. To distinguish between these possibilities, two isotopically labeled substrates (ADP-d,d-Hep) were prepared that contained 18O and 2H isotopes at C-7'' and C-6'', respectively. A crossover experiment was used to determine whether unlabeled or doubly labeled products were formed upon epimerization of a mixture of the two singly labeled compounds. After an initial epimeric equilibrium was reached, no crossover could be detected, indicating that intermediate release is not intrinsic to the overall mechanism. After extended incubation, however, scrambling of the labels could be detected, indicating that a low background rate of intermediate release does occur. To directly detect the release of the intermediate, the labeled compounds were independently epimerized in the presence of a ketone-trapping reagent, phenylhydrazine. The corresponding phenylhydrazones were identified by mass spectrometry, and the absence of any 2H isotope in the adduct obtained from the deuterated starting compound confirmed that the oxidation had occurred at C-6''.

ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the inversion of configuration at C-6' ' of the heptose moiety of ADP-d-glycero-d-manno-heptose and ADP-l-glycero-d-manno-heptose. The epimerase HldD operates in the biosynthetic pathway of l-glycero-d-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. Previous studies support a mechanism in which HldD uses its tightly bound NADP+ cofactor to oxidize directly at C-6' ', generating a ketone intermediate. A reduction of the ketone from the opposite face then occurs, generating the epimeric product. How the epimerase is able access both faces of the ketone intermediate with correct alignment of the three required components, NADPH, the ketone carbonyl, and a catalytic acid/base residue, is addressed here. It is proposed that the epimerase active site contains two catalytic pockets, each of which bears a catalytic acid/base residue that facilitates reduction of the C-6' ' ketone but leads to a distinct epimeric product. The ketone carbonyl may access either pocket via rotation about the C-5' '-C-6' ' bond of the sugar nucleotide and in doing so presents opposing faces to the bound cofactor. Evidence in support of the two-base mechanism is found in studies of two single mutants of the Escherichia coli K-12 epimerase, Y140F and K178M, both of which have severely compromised epimerase activities that are more than 3 orders of magnitude lower than that of the wild type. The catalytic competency of these two mutants in promoting redox chemistry is demonstrated with an alternate catalytic activity that requires only one catalytic base: dismutation of a C-6' ' aldehyde substrate analogue (ADP-beta-d-manno-hexodialdose) to an acid and an alcohol (ADP-beta-d-mannuronic acid and ADP-beta-d-mannose). This study identifies the two catalytic bases as tyrosine 140 and lysine 178. A one-step enzymatic conversion of mannose into ADP-beta-mannose is also described and used to make C-6' '-substituted derivatives of this sugar nucleotide.