A proposed pathway from D-glucose to D-arabinose in eukaryotes.

In eukaryotes, the D-enantiomer of arabinose (D-Ara) is an intermediate in the biosynthesis of D-erythroascorbate in yeast and fungi and in the biosynthesis of the nucleotide sugar GDP-α-D-arabinopyranose (GDP-D-Arap) and complex α-D-Arap-containing surface glycoconjugates in certain trypanosomatid parasites. Whereas the biosynthesis of D-Ara in prokaryotes is well understood, the route from D-glucose (D-Glc) to D-Ara in eukaryotes is unknown. In this paper, we study the conversion of D-Glc to D-Ara in the trypanosomatid Crithidia fasciculata using positionally labeled [13C]-D-Glc and [13C]-D-ribose ([13C]-D-Rib) precursors and a novel derivatization and gas chromatography-mass spectrometry procedure applied to a terminal metabolite, lipoarabinogalactan. These data implicate the both arms of pentose phosphate pathway and a likely role for D-ribulose-5-phosphate (D-Ru-5P) isomerization to D-Ara-5P. We tested all C. fasciculata putative sugar and polyol phosphate isomerase genes for their ability to complement a D-Ara-5P isomerase-deficient mutant of Escherichia coli and found that one, the glutamine fructose-6-phosphate aminotransferase (GFAT) of glucosamine biosynthesis, was able to rescue the E. coli mutant. We also found that GFAT genes of other trypanosomatid parasites, and those of yeast and human origin, could complement the E. coli mutant. Finally, we demonstrated biochemically that recombinant human GFAT can isomerize D-Ru-5P to D-Ara5P. From these data, we postulate a general eukaryotic pathway from D-Glc to D-Ara and discuss its possible significance. With respect to C. fasciculata, we propose that D-Ara is used not only for the synthesis of GDP-D-Arap and complex surface glycoconjugates but also in the synthesis of D-erythroascorbate.

A direct spectropolarimetric assay of arabinose 5-phosphate isomerase.

Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M-1cm-1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1-3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.

Substrate structure-activity relationship reveals a limited lipopolysaccharide chemotype range for intestinal alkaline phosphatase.

Lipopolysaccharide (LPS) from the Gram-negative bacterial outer membrane potently activates the human innate immune system. LPS is recognized by the Toll-like receptor 4/myeloid differentiation factor-2 (TLR4/MD2) complex, leading to the release of pro-inflammatory cytokines. Alkaline phosphatase (AP) is currently being investigated as an anti-inflammatory agent for detoxifying LPS through dephosphorylating lipid A, thus providing a potential treatment for managing both acute (sepsis) and chronic (metabolic endotoxemia) pathologies wherein aberrant TLR4/MD2 activation has been implicated. Endogenous LPS preparations are chemically heterogeneous, and little is known regarding the LPS chemotype substrate range of AP. Here, we investigated the activity of AP on a panel of structurally defined LPS chemotypes isolated from Escherichia coli and demonstrate that calf intestinal AP (cIAP) has only minimal activity against unmodified enteric LPS chemotypes. Pi was only released from a subset of LPS chemotypes harboring spontaneously labile phosphoethanolamine (PEtN) modifications connected through phosphoanhydride bonds. We demonstrate that the spontaneously hydrolyzed O-phosphorylethanolamine is the actual substrate for AP. We found that the 1- and 4'-lipid A phosphate groups critical in TLR4/MD2 signaling become susceptible to hydrolysis only after de-O-acylation of ester linked primary acyl chains on lipid A. Furthermore, PEtN modifications on lipid A specifically enhanced hTLR4 agonist activity of underacylated LPS preparations. Computational binding models are proposed to explain the limitation of AP substrate specificity imposed by the acylation state of lipid A, and the mechanism of PEtN in enhancing hTLR4/MD2 signaling.

Identification of a d-Arabinose-5-Phosphate Isomerase in the Gram-Positive Clostridium tetani.

d-Arabinose-5-phosphate (A5P) isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate. Various Gram-negative bacteria, such as the uropathogenic Escherichia coli strain CFT073, contain multiple API paralogs (KdsD, GutQ, KpsF, and c3406) that have been assigned various cellular functions. The d-arabinose-5-phosphate formed by these enzymes seems to play important roles in the biosynthesis of lipopolysaccharide (LPS) and group 2 K-antigen capsules, as well as in the regulation of the cellular d-glucitol uptake and uropathogenic infectivity/virulence. The genome of a Gram-positive pathogenic bacterium, Clostridium tetani, contains a gene encoding a putative API, C. tetani API (CtAPI), even though C. tetani lacks both LPS and capsid biosynthetic genes. To better understand the physiological role of d-arabinose-5-phosphate in this Gram-positive organism, recombinant CtAPI was purified and characterized. CtAPI displays biochemical characteristics similar to those of APIs from Gram-negative organisms and complements the API deficiency of an E. coli API knockout strain. Thus, CtAPI represents the first d-arabinose-5-phosphate isomerase to be identified and characterized from a Gram-positive bacterium.IMPORTANCE The genome of Clostridium tetani, a pathogenic Gram-positive bacterium and the causative agent of tetanus, contains a gene (the CtAPI gene) that shares high sequence similarity with those of genes encoding d-arabinose-5-phosphate isomerases. APIs play an important role within Gram-negative bacteria in d-arabinose-5-phosphate production for lipopolysaccharide biosynthesis, capsule formation, and regulation of cellular d-glucitol uptake. The significance of our research is in identifying and characterizing CtAPI, the first Gram-positive API. Our findings show that CtAPI is specific to the interconversion of arabinose-5-phosphate and ribulose-5-phosphate while having no activity with the other sugars and sugar phosphates tested. We have speculated a regulatory role for this API in C. tetani, an organism that does not produce lipopolysaccharide.

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.

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 novel strategy for enhancing extracellular secretion of recombinant proteins in Escherichia coli.

Secretion of cytoplasmic expressed proteins into culture medium has significant commercial advantages in large-scale production of proteins. Our previous study demonstrated that the membrane permeability of Escherichia coli could be significantly improved when Thermobifida fusca cutinase, without a signal peptide, was expressed in cytoplasm. This study investigated the extracellular production of other recombinant proteins, including both secretory and cytosolic proteins, with co-expression of cutinase. When the secretory enzymes, xylanase and α-amylase, were co-expressed with cutinase, the culture period was shortened by half, and the productivity was 7.9 and 2.0-fold to that of their individual control without co-expression, respectively. When the normally cytosolic proteins, xylose isomerase and trehalose synthase, were co-expressed with cutinase, more than half of the target proteins were "secreted" into the culture medium. Moreover, by using ß-galactosidase to detect membrane leakage, the improved secretion of the above model proteins was confirmed not to be due to cell lysis. The study provides a novel strategy for enhancing extracellular secretion of recombinant proteins in E. coli.

Extracellular location of Thermobifida fusca cutinase expressed in Escherichia coli BL21(DE3) without mediation of a signal peptide.

Cutinase is a multifunctional esterase with potential industrial applications. In the present study, a truncated version of the extracellular Thermobifida fusca cutinase without a signal peptide (referred to as cutinase(NS)) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that the majority of the cutinase activity was located in the culture medium. In a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 U/ml (2,380.8 mg/liter), and the productivity was 40.9 U/ml/h. Biochemical characterization of the purified cutinase(NS) showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinase(NS)S130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. Furthermore, T. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. Compared to the cells expressing the inactive cutinase(NS)S130A, the cells expressing cutinase(NS) showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of "cell leakage induced by the limited phospholipid hydrolysis of cutinase(NS)" was proposed to explain the underlying mechanism for the extracellular release of cutinase(NS).

Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis.

WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present crystal structures of the free and CMP-bound forms of WaaA from Aquifex aeolicus, an ancient Gram-negative hyperthermophile. These structures reveal details of the CMP-binding site and implicate a unique sequence motif (GGS/TX(5)GXNXLE) in Kdo binding. In addition, a cluster of highly conserved amino acid residues was identified which represents the potential membrane-attachment and acceptor-substrate binding site of WaaA. A series of site-directed mutagenesis experiments revealed critical roles for glycine 30 and glutamate 31 in Kdo transfer. Our results provide the structural basis of a critical reaction in LPS biosynthesis and allowed the development of a detailed model of the catalytic mechanism of WaaA.

Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3).

BACKGROUND: Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system. RESULTS: T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3). In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an α-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase. CONCLUSIONS: In the present study, T. fusca cutinase was successfully secreted to the culture media by α-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli α-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix.

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 µM, a K(M) for phosphoenolpyruvate of 891 µM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.

Evidence for a two-metal-ion mechanism in the cytidyltransferase KdsB, an enzyme involved in lipopolysaccharide biosynthesis.

Lipopolysaccharide (LPS) is located on the surface of Gram-negative bacteria and is responsible for maintaining outer membrane stability, which is a prerequisite for cell survival. Furthermore, it represents an important barrier against hostile environmental factors such as antimicrobial peptides and the complement cascade during Gram-negative infections. The sugar 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) is an integral part of LPS and plays a key role in LPS functionality. Prior to its incorporation into the LPS molecule, Kdo has to be activated by the CMP-Kdo synthetase (CKS). Based on the presence of a single Mg²âº ion in the active site, detailed models of the reaction mechanism of CKS have been developed previously. Recently, a two-metal-ion hypothesis suggested the involvement of two Mg²âº ions in Kdo activation. To further investigate the mechanistic aspects of Kdo activation, we kinetically characterized the CKS from the hyperthermophilic organism Aquifex aeolicus. In addition, we determined the crystal structure of this enzyme at a resolution of 2.10 Å and provide evidence that two Mg²âº ions are part of the active site of the enzyme.

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.

Enediol mimics as inhibitors of the D-arabinose 5-phosphate isomerase (KdsD) from Francisella tularensis.

We explored the D-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Francisella tularensis, a highly infectious gram-negative pathogen that has raised concern as a potential bioweapon, as a target for the development of novel chemotherapeutics. F. tularensis KdsD was expressed in Escherichia coli from a synthetic gene, purified, and characterized. A group of hydroxamates designed to be mimics of the putative enediol intermediate in the enzyme's catalytic mechanism were prepared and tested as inhibitors of F. tularensis KdsD. The best inhibitor, which has an IC(50) of 7 µM, is the most potent KdsD inhibitor reported to date.

The tail of KdsC: conformational changes control the activity of a haloacid dehalogenase superfamily phosphatase.

The phosphatase KdsC cleaves 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of inorganic phosphate and Kdo. Kdo is an essential component of the lipopolysaccharide envelope in Gram-negative bacteria. Because lipopolysaccharide is an important determinant of bacterial resistance and toxicity, KdsC is a potential target for novel antibacterial agents. KdsC belongs to the broad haloacid dehalogenase superfamily. In haloacid dehalogenase superfamily enzymes, substrate specificity and catalytic efficiency are generally dictated by a fold feature called the cap domain. It is therefore not clear why KdsC, which lacks a cap domain, is catalytically efficient and highly specific to 3-deoxy-D-manno-octulosonate 8-phosphate. Here, we present a set of seven structures of tetrameric Escherichia coli KdsC (ranging from 1.4 to 3.06 A in resolution) that model different intermediate states in its catalytic mechanism. A crystal structure of product-bound E. coli KdsC shows how the interface between adjacent monomers defines the active site pocket. Kdo is engaged in a network of polar and nonpolar interactions with residues at this interface, which explains substrate specificity. Furthermore, this structural and kinetic analysis strongly suggests that the binding of the flexible C-terminal region (tail) to the active site makes KdsC catalytically efficient by facilitating product release.

WaaA of the hyperthermophilic bacterium Aquifex aeolicus is a monofunctional 3-deoxy-D-manno-oct-2-ulosonic acid transferase involved in lipopolysaccharide biosynthesis.

The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli DeltawaaA suppressor strain resulted in synthesis of lipid IVA precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate.

Identification and characterization of bacterial cutinase.

Cutinase, which exists in both fungi and bacteria, catalyzes the cleavage of the ester bonds of cutin. Fungal cutinases have been extensively studied, however, reports on bacterial cutinases have been limited due to the lack of knowledge concerning the identity of their open reading frames. In the present study, the cutinase from Thermobifida fusca was induced by cutin and purified to homogeneity by following p-nitrophenyl butyrate hydrolyzing activity. Peptide mass fingerprinting analysis of the wild-type enzyme matched two proteins, Tfu_0883 and Tfu_0882, which are 93% identical in sequence. Both proteins were cloned and overexpressed in their mature form. Recombinant Tfu_0883 and Tfu_0882 display very similar enzymatic properties and were confirmed to be cutinases by their capability to hydrolyze the ester bonds of cutin. Comparative characterization of Fusarium solani pisi and T. fusca cutinases indicated that they have similar substrate specificity and catalytic properties except that the T. fusca enzymes are thermally more stable. Homology modeling revealed that T. fusca cutinases adopt an alpha/beta-hydrolase fold that exhibits both similarities and variations from the fungal cutinase structure. A serine hydrolase catalytic mechanism involving a Ser(170)-His(248)-Asp(216) (Tfu_0883 numbering) catalytic triad was supported by active site-directed inhibition studies and mutational analyses. This is the first report of cutinase encoding genes from bacterial sources.

Single amino acid substitutions in either YhjD or MsbA confer viability to 3-deoxy-d-manno-oct-2-ulosonic acid-depleted Escherichia coli.

The Escherichia coli K-12 strain KPM22, defective in synthesis of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), is viable with an outer membrane (OM) composed predominantly of lipid IV(A), a precursor of lipopolysaccharide (LPS) biosynthesis that lacks any glycosylation. To sustain viability, the presence of a second-site suppressor was proposed for transport of lipid IV(A) from the inner membrane (IM), thus relieving toxic side-effects of lipid IV(A) accumulation and providing sufficient amounts of LPS precursors to support OM biogenesis. We now report the identification of an arginine to cysteine substitution at position 134 of the conserved IM protein YhjD in KPM22 that acts as a compensatory suppressor mutation of the lethal DeltaKdo phenotype. Further, the yhjD400 suppressor allele renders the LPS transporter MsbA dispensable for lipid IV(A) transmembrane trafficking. The independent derivation of a series of non-conditional KPM22-like mutants from the Kdo-dependent parent strain TCM15 revealed a second class of suppressor mutations localized to MsbA. Proline to serine substitutions at either residue 18 or 50 of MsbA relieved the Kdo growth dependence observed in the isogenic wild-type strain. The possible impact of these suppressor mutations on structure and function are discussed by means of a computationally derived threading model of MsbA.