Bacterial 1,4-α-glucan branching enzymes: characteristics, preparation and commercial applications.

The 1,4-α-glucan branching enzymes (GBEs) are ubiquitously distributed in animals, microorganisms and plants. These enzymes modify the structure of both starch and glycogen; changing the frequency and position of branches by forming new α-1,6-glucosidic linkages. In organisms, controlling the number and distribution of branches is an irreplaceable process that maintains the physiological state of starch and glycogen in the cell. The process is also the foundation for the industrial applications of GBEs. So far, a number of GBEs have been identified in eukaryotes and prokaryotes as researchers searched for GBEs with optimal properties. Among them, bacterial GBEs have received particular attention due to the convenience of heterologous expression and industrial applications of GBEs from bacteria than GBEs from other sources. The advantages of bacterial GBEs in potential applications stimulated the investigations of bacterial GBEs in terms of their structure and properties. However, full exploitation of GBEs in commercial applications is still in its infancy because of the disadvantages of currently available enzymes and of limited imagination with respect to future possibilities. Thus, in this review, we present an overview of the bacterial GBEs including their structure, biochemical properties and commercial applications in order to depict the whole picture of bacterial GBEs.

Leu600 mutations decrease product inhibition of the ß-cyclodextrin glycosyltransferase from Bacillus circulans STB01.

The limits that cyclodextrin products impose on their industrial production from starch by cyclodextrin glycosyltransferases (CGTases) are a severe problem. In this paper, mutants at residue Leu600 of the ß-CGTase from Bacillus circulans STB01 were constructed in an effort to decrease the product inhibition exhibited by ß-cyclodextrin. A kinetic analysis of the inhibition of the wild-type and mutant ß-CGTases by ß-cyclodextrin revealed that the mutations do not alter the inhibition mode, which is mixed-type. However, the values of the inhibition constants (Ki and Ki') of the mutants L600I, L600E and L600R are higher than those of the wild-type enzyme, weakening the product inhibition. The mutant L600Y only exhibited a decrease in noncompetitive inhibition, with the value of Ki' increasing by 40%. Comparison of the Km' values and the 3D model structures of the wild-type and mutant CGTases suggests that this decrease in product inhibition is related to a decrease in binding affinity between the product cyclodextrin and the enzyme.

Maltooligosaccharide-forming amylase: Characteristics, preparation, and application.

As member of glycosyl hydrolase family 13, maltooligosaccharide-forming amylases (MFAses) are specific and interesting because of their capacity to hydrolyze starch into functional maltooligosaccharides, which are usually composed of 2-10 α-d-glucopyranosyl units linked by α-1,4 glycosidic linkages. MFAses have been extensively studied during recent decades, and have shown promise in various industrial applications. This review begins by introducing the potential uses of maltooligosaccharides. Then it describes the progress in the identification, assay, action pattern, structure, and modification of MFAses. The review continues with tips concerning the preparation of MFAses, which aim to improve MFAse production to meet the needs of industry. Finally, the industrial uses of MFAses are described, focusing on the production of maltooligosaccharides and application in the bread industry. Recent progress has demonstrated that the MFAses are poised to become important industrial catalysts.

Asp577 mutations enhance the catalytic efficiency of cyclodextrin glycosyltransferase from Bacillus circulans.

The amino acid residue Asp 577 is located in calcium-binding site III (CaIII) of the cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) from Bacillus circulans STB01. In the present study, the effects of replacing Asp577 with glycine, alanine, valine, leucine, and isoleucine on the catalytic efficiency of this CGTase were investigated. Two of these replacements, D577G and D577A, increased the ß-cyclization activity of CGTase. Kinetic studies showed that the Km values of D577G and D577A were 36.1% and 18.0% lower and the kcat/Km values were 43.9% and 23.0% higher than those of the wild-type enzyme, respectively. These mutations increased both the affinity of CGTase for maltodextrin and the catalytic efficiency of the cyclization reaction. Furthermore, although D577G and D577A only slightly enhanced ß-cyclodextrin production, compared with the wild-type enzyme, their higher ß-cyclization activities resulted in a significant reduction in the amount of mutant protein required during the cyclodextrin production process. Thus, the two mutants are more suitable for the industrial production of ß-cyclodextrin than the wild-type enzyme. The enhancement of catalytic efficiency may be due to the smaller size of the glycine and alanine side chains, which may weaken the impact of this residue on CaIII.

A novel glucose 6-phosphate isomerase from Listeria monocytogenes.

D-Arabinose 5-phosphate isomerases (APIs) catalyze the interconversion of D-ribulose 5-phosphate and D-arabinose 5-phosphate (A5P). A5P is an intermediate in the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo), an essential component of lipopolysaccharide, the lipopolysaccharide found in the outer membrane of Gram-negative bacteria. The genome of the Gram-positive pathogen Listeria monocytogenes contains a gene encoding a putative sugar isomerase domain API, Q723E8, with significant similarity to c3406, the only one of four APIs from Escherichia coli CFT073 that lacks a cystathionine-ß-synthase domain. However, L. monocytogenes lacks genes encoding any of the other enzymes of the Kdo biosynthesis pathway. Realizing that the discovery of an API in a Gram-positive bacterium could provide insight into an alternate physiological role of A5P in the cell, we prepared and purified recombinant Q723E8. We found that Q723E8 does not possess API activity, but instead is a novel GPI (D-glucose 6-phosphate isomerase). However, the GPI activity of Q723E8 is weak compared with previously described GPIS. L. monocytogenes contains an ortholog of the well-studied two-domain bacterial GPI, so this maybe redundant. Based on this evidence glucose utilization is likely not the primary physiological role of Q723E8.

Polyethylene glycols enhance the thermostability of ß-cyclodextrin glycosyltransferase from Bacillus circulans.

We investigated the ability of six polyethylene glycols (PEGs), with molecular weights ranging from 400 to 20,000 Da, to enhance the thermostability of ß-cyclodextrin glycosyltransferase (ß-CGTase) from Bacillus circulans. We found that PEGs with different molecular weights could activate and stabilize this ß-CGTase, but to different degrees. The most significant increase (about 20%) in ß-cyclodextrin-forming activity was achieved by adding 10-15% PEG 400. PEGs with low molecular weights also significantly enhanced the thermostability of ß-CGTase; 15% PEG 1000 prolonged its half-life at 60°C by 6.5-fold, compared to a control. Fluorescence spectroscopy and circular dichroism analysis indicated that PEGs helped protect the tertiary and secondary structure of ß-CGTase, respectively. This study provides an effective approach for improving the thermostability of CGTases and related enzymes.

Analysis of the arabinose-5-phosphate isomerase of Bacteroides fragilis provides insight into regulation of single-domain arabinose phosphate isomerases.

Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and D-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-D-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant Q5LIW1 protein (here referred to by the UniProt ID Q5LIW1), characterizing its API activity in vitro, and demonstrating that the gene encoding Q5LIW1 (GenBank ID YP_209877.1) was able to complement an API-deficient E. coli strain. We demonstrated that Q5LIW1 is inhibited by cytidine 5'-monophospho-3-deoxy-D-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a Ki of 1.91 µM. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that Q5LIW1 may be a suitable system to study API structure-function relationships.

Arabinose 5-phosphate isomerase as a target for antibacterial design: studies with substrate analogues and inhibitors.

Structural requirements of D-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Pseudomonas aeruginosa were analysed in detail using advanced NMR techniques. We performed epitope mapping studies of the binding between the enzyme and the most potent KdsD inhibitors found to date, together with studies of a set of newly synthesised arabinose 5-phosphate (A5P) mimetics. We report here the first experimental evidence that KdsD may bind the furanose form of A5P, suggesting that catalysis of ring opening may be an important part of KdsD catalysis.

A simple assay for 3-deoxy-d-manno-octulosonate cytidylyltransferase and its use as a pathway screen.

This article describes the adaptation of a simple colorimetric assay for inorganic pyrophosphate to the enzyme 3-deoxy-d-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase, KdsB, EC 2.7.7.38), a key enzyme in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative organisms. This assay is particularly useful because it can be combined with the malachite green (MG) assay for inorganic phosphate to form an assay system capable of determining inorganic phosphate and inorganic pyrophosphate in the same solution (the MG/EK (eikonogen reagent) assay). This assay system has the potential for simultaneous screening of the 3-deoxy-d-manno-octulosonate (KDO) biosynthesis pathway. We tested this potential using two enzymes, KdsB and KdsC, involved in the biosynthesis and use of the key bacterial 8-carbon sugar, KDO.

A unique arabinose 5-phosphate isomerase found within a genomic island associated with the uropathogenicity of Escherichia coli CFT073.

Previous studies showed that deletion of genes c3405 to c3410 from PAI-metV, a genomic island from Escherichia coli CFT073, results in a strain that fails to compete with wild-type CFT073 after a transurethral cochallenge in mice and is deficient in the ability to independently colonize the mouse kidney. Our analysis of c3405 to c3410 suggests that these genes constitute an operon with a role in the internalization and utilization of an unknown carbohydrate. This operon is not found in E. coli K-12 but is present in a small number of pathogenic E. coli and Shigella boydii strains. One of the genes, c3406, encodes a protein with significant homology to the sugar isomerase domain of arabinose 5-phosphate isomerases but lacking the tandem cystathionine beta-synthase domains found in the other arabinose 5-phosphate isomerases of E. coli. We prepared recombinant c3406 protein, found it to possess arabinose 5-phosphate isomerase activity, and characterized this activity in detail. We also constructed a c3406 deletion mutant of E. coli CFT073 and demonstrated that this deletion mutant was still able to compete with wild-type CFT073 in a transurethral cochallenge in mice and could colonize the mouse kidney. These results demonstrate that the presence of c3406 is not essential for a pathogenic phenotype.

Trypanosoma brucei glycogen synthase kinase-3, a target for anti-trypanosomal drug development: a public-private partnership to identify novel leads.

BACKGROUND: Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3ß (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT. METHODOLOGY/PRINCIPAL FINDINGS: A subset of over 16,000 inhibitors of HsGSK-3 ß from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3ß and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed. CONCLUSIONS/SIGNIFICANCE: We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds.

Targeting the non-structural proteins of hepatitis C virus: beyond hepatitis C virus protease and polymerase.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is a main cause of cirrhosis of the liver and hepatocellular carcinoma. The standard of care is a combination of pegylated interferon with ribavirin, a regimen that has undesirable side effects and is frequently ineffective. Compounds targeting HCV protease and polymerase are in late-stage clinical trials and have been extensively reviewed elsewhere. OBJECTIVE: To review and evaluate the progress towards finding novel HCV antivirals targeting HCV proteins beyond the already precedented NS3 protease and NS5B polymerase. METHODS: Searches of CAplus and Medline databases were combined with information from key conferences. This review focuses on NS2/3 serine protease, NS3 helicase activity and the non-structural proteins 4A, 4B and 5A. CONCLUSIONS: Use of the replicon model of HCV replication and biochemical assays of specific targets has allowed screening of vast libraries of compounds, but resulted in clinical candidates from only NS4A and NS5A. The field is hindered by a lack of good chemical matter that inhibits the remaining enzymes from HCV, and a lack of understanding of the functions of non-structural proteins 4A, 4B and 5A in the replication of HCV.

Rational protein engineering in action: the first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus.

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

Simultaneous screening of multiple bacterial tRNA synthetases using an Escherichia coli S30-based transcription and translation assay.

The search for novel antibiotics to combat the growing threat of resistance has led researchers to screen libraries with coupled transcription and translation systems. In these systems, a bacterial cell lysate supplies the proteins necessary for transcription and translation, a plasmid encoding a reporter protein is added as a template, and a complex mixture of amino acids and cofactors is added to supply building blocks and energy to the assay. Firefly luciferase is typically used as the reporter protein in high-throughput screens because the luminescent signal is strong and, since bacterial lysates contain no luciferase, the background is negligible. The typical coupled transcription and translation assay is sensitive to inhibitors of RNA polymerase and to compounds that bind tightly to the ribosome. We have found a way to increase the information content of the screen by making the assay more sensitive to inhibitors of tRNA synthetases. Restricting the concentration of amino acids added to the reaction mixture allows the simultaneous screening of multiple tRNA synthetase enzymes along with the classic transcription and translation targets. In addition, this assay can be used as a convenient way to determine if an antibacterial compound of unknown mechanism inhibits translation through inhibition of a tRNA synthetase, and to identify which synthetase is the target.

Structural biology approaches to antibacterial drug discovery.

Antibacterial drug discovery has undertaken a major experiment in the 12 years since the first bacterial genomes were sequenced. Genome mining has identified hundreds of potential targets that have been distilled to a relatively small number of broad-spectrum targets ('low-hanging fruit') using the genetics tools of modern microbiology. Prosecuting these targets with high-throughput screens has led to a disappointingly small number of lead series that have mostly evaporated under closer scrutiny. In the meantime, multi-drug resistant pathogens are becoming a serious challenge in the clinic and the community and the number of pharmaceutical firms pursuing antibacterial discovery has declined. Filling the antibacterial development pipeline with novel chemical series is a significant challenge that will require the collaboration of scientists from many disciplines. Fortunately, advancements in the tools of structural biology and of in silico modeling are opening up new avenues of research that may help deal with the problems associated with discovering novel antibiotics.

In vivo labeling: a glimpse of the dynamic proteome and additional constraints for protein identification.

Identities ascribed to the intact protein ions detected in MALDI-MS of whole bacterial cells or from other complex mixtures are often ambiguous. Isolation of candidate proteins can establish that they are of correct molecular mass and sufficiently abundant, but by itself is not definitive. An in vivo labeling strategy replacing methionine with selenomethionine has been employed to deliver an additional constraint for protein identification, i.e., number of methionine residues, derived from the shift in mass of labeled versus unlabeled proteins. By stressing a culture and simultaneously labeling, it was possible to specifically image the cells' response to the perturbation. Because labeled protein is only synthesized after application of the stress, it provides a means to view dynamic changes in the cellular proteome. These methods have been applied to identify a 15,879 Da protein ion from E. coli that was induced by an antibacterial agent with an unknown mechanism of action as SpY, a stress protein produced abundantly in spheroplasts. It has also allowed us to propose protein identities (and eliminate others from consideration) for many of the ions observed in MALDI (and ESI-MS) whole cell profiling at a specified growth condition.

Synthesis and structure-activity relationship study of potent trypanocidal thio semicarbazone inhibitors of the trypanosomal cysteine protease cruzain.

American trypanosomiasis, or Chagas' disease, is the leading cause of heart disease in Latin America. Currently there is an urgent need to develop antitrypanosomal therapy due to the toxicity of existing agents and emerging drug resistance. A novel series of potent thio semicarbazone small-molecule inhibitors of the Trypanosoma cruzi cysteine protease cruzain have been identified. Some of these inhibitors have been shown to be trypanocidal. We initially discovered that 3'-bromopropiophenone thio semicarbazone (1i) inhibited cruzain and could cure mammalian cell cultures infected with T. cruzi. 3'-Bromopropiophenone thio semicarbazone showed no toxicity for mammalian cells at concentrations that were trypanocidal. Following this lead, more than 100 compounds were designed and synthesized. A specific structure-activity relationship (SAR) was established, and many potent analogues with IC(50) values in the low nanomolar range were identified. Eight additional analogues were trypanocidal in a cell culture assay, and this indicates that aryl thio semicarbazone is a productive scaffold for killing the parasites. Kinetic studies show that these are time-dependent inhibitors. Molecular modeling studies of the enzyme-inhibitor complex have led to a proposed mechanism of interaction as well as insight into the SAR of the thio semicarbazone series. The nonpeptide nature of this series, small size, and extremely low cost of production suggest this is a promising direction for the development of new antitrypanosome chemotherapy.