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PubMed1995

Crystallization and preliminary crystallographic data for class I deoxyribose-5-phosphate aldolase from Escherichia coli: an application of reverse screening.

X-ray quality crystals of class I-deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate. The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 183.1 A, b = 61.4 A, c = 49.3 A and a = 179.2 A, b = 60.5, A, c = 49.1 A, respectively. Two molecules in the asymmetric unit are related by a noncrystallographic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 A. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions.

Aldehyde-Lyases↗
PubMed1999

The phnIJ genes encoding acetaldehyde dehydrogenase (acylating) and 4-hydroxy-2-oxovalerate aldolase in Pseudomonas sp. DJ77 and their evolutionary implications.

The two final steps of meta-cleavage pathway for catechol degradation involve conversion of 4-hydroxy-2-oxovalerate, via acetaldehyde, to acetyl coenzyme A. We report here the complete nucleotide sequences and overexpression of the phnIJ genes for an acetaldehyde dehydrogenase (acylating) (ADA) and a 4-hydroxy-2-oxovalerate aldolase (HOA) from the meta-pathway operon of the phenanthrene-degrading bacterium, Pseudomonas sp. strain DJ77. Additional partial sequence analysis of adjacent DNA shows the gene order within the operon to be phnHIJ, identical to the order found for the isofunctional genes in the other meta-pathway operons. The deduced amino acid sequences of the PhnI (312 amino acids) and PhnJ (343 amino acids) have identities of 51-71% with the corresponding genes of dmp, xyl, nah, bph_LB400, bph_KKS102, tod, cum, cmt, and MTCY03C7 operons. The phylogenetic analyses reveal the evolutionary relationships of HOA and ADA.

Acetaldehyde↗
PubMed2000

Structures of l-fuculose-1-phosphate aldolase mutants outlining motions during catalysis.

The crystal structures of l-fuculose-1-phosphate aldolase (FucA) with and without a ligated analogue of dihydroxyacetone phosphate (DHAP) and of a number of active center mutants have resulted in a model of the catalytic mechanism. This model has now been confirmed by structural analyses of further mutations at the zinc coordination sphere and at the phosphate site. In addition, these mutants have revealed new aspects of the catalysis: the hydroxyl group of Tyr113' (from a neighboring subunit), which sits just outside the zinc coordination sphere, steers DHAP towards a productive binding mode at the zinc ion; Glu73 contacts zinc in between the two ligand positions intended for the DHAP oxygen atoms and thus avoids blocking of these positions by a tetrahedrally coordinated hydroxy ion; the FucA polypeptide does not assume its minimum energy state but oscillates between two states of elevated energy as demonstrated by a mutant in a minimum energy state. The back and forth motion involves a mobile loop connecting the phosphate site with intersubunit motions and thus with the Brownian motion of the solvent. The phosphate group is bound strongly at a given distance to the zinc ion, which prevents the formation of too tight a DHAP:zinc complex. This observation explains our failure to find mutants that accept phosphate-free substitutes for DHAP. The FucA zinc coordination sphere is compared with that of carbonic anhydrase.

Aldehyde-Lyases↗
PubMed1999

A new route to L-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity D-threonine aldolase-catalyzed stereospecific resolution.

A new enzymatic resolution process was established for the production of L-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity D-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized DL-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichia coli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l-1) L-threo-MTPS was obtained from 200 mM (45.5 g l-1) DL-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess.

Anti-Bacterial Agents↗
PubMed1984

Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis.

2-Keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) has been isolated from extracts of Zymomonas mobilis using differential dye-ligand chromatography and affinity elution with product/product analog. The one-step procedure gives an enzyme with specific activity 600 units mg-1. Only 1 out of 47 dyes, Procion Yellow MX-GR, bound the enzyme completely in 20 mM phosphate buffer, pH 6.5. A column of Navy HE-R adsorbent was used first to remove most of the potentially adsorbing proteins.

Adsorption↗
PubMed1979

Tritium exchange reactions catalyzed by 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli K-12.

Tritiated water and tritiated substrates have been used to study exchange reactions catalyzed by Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16, 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate). With pyruvate, the enzyme catalyzes a rapid first-order exchange of all three methyl hydrogens in the absence of added acceptor aldehyde (i.e. glyoxylate). This reaction is not rate limiting for aldol condensation or cleavage; quite different pH-activity profiles for the exchange reaction versus aldol cleavage and also comparative effects that pH changes have on Km and V values for the two processes favor this conclusion. The exchange reaction with 2-oxobutyrate, a substrate analog, is stereoselective; one methylene hydrogen is removed at a 6-fold faster rate than the other but eventually both are exchanged. No tritium exchange occurs with glyoxylate.

Escherichia coli↗
PubMed1985

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid.

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1995

Two domains with amino-acid sequence similarity are required for dihydroneopterin aldolase function in the multifunctional folic acid synthesis Fas protein of Pneumocystis carinii.

The folic acid synthesized gene (fas) of Pneumocystis carinii (Pc) codes for a multifunctional enzyme (Fas) known to catalyse three consecutive steps leading to the production of dihydropteroate in the de novo folate synthesis pathway. Previously, we predicted that a domain, designated FasB (amino acids (aa) 161-280), of the 740-aa multifunctional protein contains the first of the three enzyme activities in the pathway, namely dihydroneopterin aldolase (DHNA), since it shares 23% aa identity with the DHNA of Streptococcus pneumoniae (Sp). We now extend these findings to show that a second domain, FasA (aa 39-160), whose function was previously unknown, shares 27% sequence identity with the adjacent FasB domain, indicative of functional similarity. FasA is also 18% identical with the DHNA from Sp. Recombinant baculoviruses were constructed which directed the production of either FasA, FasB or FasAB polypeptide species in cultured Spodoptera frugiperda (SF9) insect cells. No DHNA activity is associated with either fasA or fasB when produced as single domains in the insect-baculovirus system. However, DHNA activity was detected in SF9 extracts containing the overproduced FasAB polypeptide. The results of aa sequence alignments and expression studies suggest that FasA and FasB may be two subunits of the DHNA enzyme moiety within the multifunctional Fas protein of Pc. An alternative interpretation of the results is also discussed.

Aldehyde-Lyases↗
PubMed2006

Cell permeation of a Trypanosoma brucei aldolase inhibitor: evaluation of different enzyme-labile phosphate protecting groups.

A series of four prodrugs directed against Trypanosoma brucei aldolase bearing various transient enzyme-labile phosphate protecting groups was developed. Herein, we describe the synthesis and evaluation of cell permeation of these prodrugs. The oxymethyl derivative was the most efficient prodrug with a good recovering of the free drug (IC(50)=20 microM) and without any measurable cytotoxicity.

Aldehyde-Lyases↗
PubMed2005

Influence of induction and operation mode on recombinant rhamnulose 1-phosphate aldolase production by Escherichia coli using the T5 promoter.

Fed-batch culture techniques were employed to grow Escherichia coli at high-cell densities for the intracellular production of a recombinant rhamnulose 1-phosphate aldolase (RhuA) under the transcriptional control of the strong promoter T5, using a commercially available expression system (E. coli strain M15 and plasmid vector pQE40). A predetermined exponential feeding strategy at constant specific growth rate was selected to maintain carbon source limited growth using a defined medium. Growth rates below 0.36 h(-1) did not cause a severe formation of acetic acid while cell concentration increased exponentially up to 95 g(biomass)l(-1). The intrinsic biomass-substrate yield (gamma(X/S) = 0.48 gg(-1)) and the maintenance coefficient (mS = 0.10 g(substrate)g(biomass)(-1)h(-1)) were calculated from fed-batch growths at different specific growth rates. These values have been employed to determinate the addition profile during the fed-batch growth until IPTG induction, reaching a specific RhuA production levels of 565 AU g(biomass)(-1) which was lower than in batch (1250 AU g(biomass)(-1)). An inverse correlation between volumetric IPTG concentration and specific RhuA activity was found. A correlation between the ratio biomass/OD(600 nm) and the quantity of recombinant protein produced was found. Finally, the proposed process, after optimization of the IPTG concentration, led to significant increase in enzyme concentration and volumetric productivity compared to batch mode (2680 and 1338%, respectively).

Aldehyde-Lyases↗
PubMed2005

Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16.

Two different recombinant plasmids both containing the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 but differing concerning the resistance marker gene were tested for their suitability to produce high amounts of cyanophycin in recombinant strains of Ralstonia eutropha. Various cultivation experiments at the 30-L scale revealed very low cyanophycin contents of the cells ranging from 4.6% to 6.2% (w/w) of cellular dry weight (CDW) only, most probably because most cells had lost the corresponding plasmid during cultivation. To establish a cost effective and high efficient system for production of cyanophycin at larger scales using recombinant strains of R. eutropha, we applied two strategies: First, we integrated cphA into the dispensable chromosomal l-lactate dehydrogenase gene (ldh) of R. eutropha. Depending on the cultivation conditions used, relatively low cyanophycin contents between 2.2% and 7.7% (w/w) of CDW were reproducibly detected, which might be due to weak expression or low gene dosage in the single cphA copy strain of R. eutropha. In a second strategy we constructed a KDPG-aldolase gene (eda)-dependent addiction system, which combined features of a multi-copy plasmid with stabilized expression of cphA. Flasks experiments revealed that the cells accumulated extraordinarily high amounts of cyanophycin between 26.9% and 40.0% (w/w) of CDW even under cultivation conditions lacking cyanophycin precursor substrates or plasmid stabilizing antibiotics. Cyanophycin contents of up to 40.0% (w/w) of CDW were also obtained at a 30-L scale or a 500-L pilot-plant scale under such non-selective conditions. This demonstrates impressively that the stabilizing effect of the constructed eda-dependent addiction system can be used for production of enhanced amounts of cyanophycin at a larger scale in recombinant strains of R. eutropha.

Aldehyde-Lyases↗
PubMed2001

Synthesis of C-3 nitrogen-containing derivatives of N-acetyl-alpha,beta-D-mannosamine as substrates for N-acetylneuraminic acid aldolase.

The synthesis of 3-azido-3-deoxy, 3-amino-3-deoxy and 3-N-tert-butyloxycarbonyl-3-deoxy derivatives of 2-acetamido-2-deoxy-alpha,beta-D-mannose (N-acetyl-alpha,beta-D-mannosamine, ManNAc), is presented. The 3-azido-3-deoxy- and 3-N-tert-butyloxycarbonyl compounds were further characterised as their peracetates. A preliminary study has found that these C-3 nitrogen-substituted derivatives of ManNAc not to be substrates for Neu5Ac aldolase.

Hexosamines↗
PubMed2001

Biocatalytic synthesis of hydroxylated natural products using aldolases and related enzymes.

Synthetic building blocks bearing hydroxylated chiral centers are important targets for biocatalysis. Many C-C bond forming enzymes have recently been investigated for new applications and new strategies towards the synthesis of natural products and related oxygenated compounds. Several old catalysts have been studied to increase our functional knowledge of natural aldolase-type enzymes, and new mutated catalysts or catalytic antibodies have been tested for their synthetic utility.

Aldehyde-Lyases↗
PubMed2003

Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions.

Expanding the scope of substrate specificity and stereoselectivity is of current interest in enzyme catalysis. Using error-prone PCR for in vitro directed evolution, the Neu5Ac aldolase from Escherichia coli has been altered to improve its catalytic activity toward enantiomeric substrates including N-acetyl-L-mannosamine and L-arabinose to produce L-sialic acid and L-KDO, the mirror-image sugars of the corresponding naturally occurring D-sugars. The first generation variant containing two mutations (Tyr98His and Phe115Leu) outside the (alpha,beta)(8)-barrel active site exhibits an inversion of enantioselectivity toward KDO and the second generation variant contains an additional amino acid change Val251Ile outside the alpha,beta-barrel active site that improves the enantiomeric formation of L-sialic acid and L-KDO. The X-ray structure of the triple mutant epNanA.2.5 at 2.3A resolution showed no significant difference between the wild-type and the mutant enzymes. We probed the potential structural 'hot spot' of enantioselectivity with saturation mutagenesis at Val251, the mutated residue most proximal to the Schiff base forming Lys165. The selected variant had an increase in k(cat) via replacement with another hydrophobic residue, leucine. Further sampling of a larger sequence space with error-prone PCR selected a third generation variant with significant improvement in L-KDO catalysis and a complete reversal of enantioselectivity.

Aldehydes↗
PubMed2000

Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase.

Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase. Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. 1H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme.

Bacterial Proteins↗
PubMed2000

Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase.

A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.

Aldehyde-Lyases↗
PubMed2002

X-ray structures of threonine aldolase complexes: structural basis of substrate recognition.

L-Threonine acetaldehyde-lyase (threonine aldolase, TA) is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes conversion of L-threonine or L-allo-threonine to glycine and acetaldehyde in a secondary glycine biosynthetic pathway. X-ray structures of Thermatoga maritima TA have been determined as the apo-enzyme at 1.8 A resolution and bound to substrate L-allo-threonine and product glycine at 1.9 and 2.0 A resolution, respectively. Despite low pairwise sequence identities, TA is a member of aspartate aminotransferase (AATase) fold family of PLP enzymes. The enzyme forms a 222 homotetramer with the PLP cofactor bound via a Schiff-base linkage to Lys199 within a domain interface. The structure reveals bound calcium and chloride ions that appear to contribute to catalysis and oligomerization, respectively. Although L-threonine and L-allo-threonine are substrates for T. maritima TA, enzymatic assays revealed a strong preference for L-allo-threonine. Structures of the external aldimines with substrate/product reveal a pair of histidines that may provide flexibility in substrate recognition. Variation in the threonine binding pocket may explain preferences for L-allo-threonine versus L-threonine among TA family members.

Amino Acid Sequence↗