Purification and characterization of the class-II D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes' strain).
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ABSTRACT:
SUBMITTER: Baldwin SA
PROVIDER: S-EPMC1183836 | biostudies-other | 1978 Mar
REPOSITORIES: biostudies-other
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Project description:Nickel is toxic to all forms of life, but the mechanisms of cell damage are unknown. Indeed, environmentally relevant nickel levels (8 µM) inhibit wild-type Escherichia coli growth on glucose minimal medium. The same concentration of nickel also inhibits growth on fructose, but not succinate, lactate or glycerol; these results suggest that fructose-1,6-bisphosphate aldolase (FbaA) is a target of nickel toxicity. Cells stressed by 8 µM Ni(II) for 20 min lost 75% of their FbaA activity, demonstrating that FbaA is inactivated during nickel stress. Furthermore, overexpression of fbaA restored growth of an rcnA mutant in glucose minimal medium supplemented with 4 µM Ni(II), thus confirming that FbaA is a primary target of nickel toxicity. This class II aldolase has an active site zinc and a non-catalytic zinc nearby. Purified FbaA lost 80 % of its activity within 2 min when challenged with 8 µM Ni(II). Nickel-challenged FbaA lost 0.8 zinc and gained 0.8 nickel per inactivated monomer. FbaA mutants (D144A and E174A) affecting the non-catalytic zinc were resistant to nickel inhibition. These results define the primary site of nickel toxicity in E. coli as the class II aldolase FbaA through binding to the non-catalytic zinc site.
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Project description:The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBanktrade mark database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies.
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Project description:Class II fructose 1,6-bisphosphate aldolase (FBA) is an enzyme critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis. Importantly, humans lack this type of aldolase, having instead a class I FBA that is structurally and mechanistically distinct from class II FBAs. As such, class II FBA is considered a putative pharmacological target for the development of novel antibiotics against pathogenic bacteria such as Mycobacterium tuberculosis, the causative agent for tuberculosis (TB). To date, several competitive class II FBA substrate mimic-styled inhibitors have been developed; however, they lack either specificity, potency, or properties that limit their potential as possible therapeutics. Recently, through the use of enzymatic and structure-based assisted screening, we identified 8-hydroxyquinoline carboxylic acid (HCA) that has an IC50 of 10 ± 1 ?M for the class II FBA present in M. tuberculosis (MtFBA). As opposed to previous inhibitors, HCA behaves in a noncompetitive manner, shows no inhibitory properties toward human and rabbit class I FBAs, and possesses anti-TB properties. Furthermore, we were able to determine the crystal structure of HCA bound to MtFBA to 2.1 Å. HCA also demonstrates inhibitory effects for other class II FBAs, including pathogenic bacteria such as methicillin-resistant Staphylococcus aureus. With its broad-spectrum potential, unique inhibitory characteristics, and flexibility of functionalization, the HCA scaffold likely represents an important advancement in the development of class II FBA inhibitors that can serve as viable preclinical candidates.
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Project description:Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=72.39, b=127.71, c=157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site.
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Project description:The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolase has been crystallized in space group P22121, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0 Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.
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Project description:Investigation of aldolase 1, the class-I D-fructose 1,6-bisphosphate aldolase (EC4.1.2.13) from Escherichia coli (Crookes' strain), showed it to have unusual kinetic and structural properties. The enzyme appeared to be larger than was previously supposed and may be a decamer with a mol. wt. of approx. 340000. Its fructose 1,6-bisphosphate-cleavage activity was unaffected by these compounds. The enhancement exhibited a strong dependence on pH. These novel kinetic properties do not seem to be shared by any other fructose 1,6-bisphosphate aldolase, but recall the activation by polycarboxylic acids of the deoxyribose 3-phosphate aldolases from some other organisms. In view of its unusual properties, it is unlikely that aldolase 1 from E. coli is closely related to the class-1 aldolases that have been detected in several other prokaryotes, or to the typical class-1 enzymes from eukaryotes.
| S-EPMC1183837 | biostudies-other
Project description:Fructose 1,6-bisphosphate aldolase is a ubiquitous cytosolic enzyme that catalyzes the fourth step of glycolysis. Aldolases are classified into three groups: Class-I, Class-IA, and Class-II; all classes share similar structural features but low amino acid identity. Apart from their conserved role in carbohydrate metabolism, aldolases have been reported to perform numerous non-enzymatic functions. Here we review the myriad "moonlighting" functions of this classical enzyme, many of which are centered on its ability to bind to an array of partner proteins that impact cellular scaffolding, signaling, transcription, and motility. In addition to the cytosolic location, aldolase has been found the extracellular surface of several pathogenic bacteria, fungi, protozoans, and metazoans. In the extracellular space, the enzyme has been reported to perform virulence-enhancing moonlighting functions e.g., plasminogen binding, host cell adhesion, and immunomodulation. Aldolase's importance has made it both a drug target and vaccine candidate. In this review, we note the several inhibitors that have been synthesized with high specificity for the aldolases of pathogens and cancer cells and have been shown to inhibit classical enzyme activity and moonlighting functions. We also review the many trials in which recombinant aldolases have been used as vaccine targets against a wide variety of pathogenic organisms including bacteria, fungi, and metazoan parasites. Most of such trials generated significant protection from challenge infection, correlated with antigen-specific cellular and humoral immune responses. We argue that refinement of aldolase antigen preparations and expansion of immunization trials should be encouraged to promote the advancement of promising, protective aldolase vaccines.
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Project description:Fructose 1,6-bisphosphate aldolase (FBA) is important for both glycolysis and gluconeogenesis in life. Class II (zinc dependent) FBA is an attractive target for the development of antibiotics against protozoa, bacteria, and fungi, and is also widely used to produce various high-value stereoisomers in the chemical and pharmaceutical industry. In this study, the crystal structures of class II Escherichia coli FBA (EcFBA) were determined from four different crystals, with resolutions between 1.8 Å and 2.0 Å. Native EcFBA structures showed two separate sites of Zn1 (interior position) and Zn2 (active site surface position) for Zn2＋ ion. Citrate and TRIS bound EcFBA structures showed Zn2＋ position exclusively at Zn2. Crystallographic snapshots of EcFBA structures with and without ligand binding proposed the rationale of metal shift at the active site, which might be a hidden mechanism to keep the trace metal cofactor Zn2＋ within EcFBA without losing it. [BMB Reports 2016; 49(12): 681-686].
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Project description:Class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff base intermediate using an ?-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation-deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation-protonation step of the MtFBA reaction mechanism. Also, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.
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Project description:Staphylococcus aureus is one of the most common nosocomial sources of soft-tissue and skin infections and has more recently become prevalent in the community setting as well. Since the use of penicillins to combat S. aureus infections in the 1940s, the bacterium has been notorious for developing resistances to antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA). With the persistence of MRSA as well as many other drug resistant bacteria and parasites, there is a growing need to focus on new pharmacological targets. Recently, class II fructose 1,6-bisphosphate aldolases (FBAs) have garnered attention to fill this role. Regrettably, scarce biochemical data and no structural data are currently available for the class II FBA found in MRSA (SaFBA). With the recent finding of a flexible active site zinc-binding loop (Z-Loop) in class IIa FBAs and its potential for broad spectrum class II FBA inhibition, the lack of information regarding this feature of class IIb FBAs, such as SaFBA, has been limiting for further Z-loop inhibitor development. Therefore, we elucidated the crystal structure of SaFBA to 2.1 Å allowing for a more direct structural analysis of SaFBA. Furthermore, we determined the KM for one of SaFBA's substrates, fructose 1,6-bisphosphate, as well as performed mode of inhibition studies for an inhibitor that takes advantage of the Z-loop's flexibility. Together the data offers insight into a class IIb FBA from a pervasively drug resistant bacterium and a comparison of Z-loops and other features between the different subtypes of class II FBAs.
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