Α-ketoglutarate Semialdehyde Research Articles

Enzyme activity engineering based on sequence co-evolution analysis

The utility of engineering enzyme activity is expanding with the development of biotechnology. Conventional methods have limited applicability as they require high-throughput screening or three-dimensional structures to direct target residues of activity control. An alternative method uses sequence evolution of natural selection. A repertoire of mutations was selected for fine-tuning enzyme activities to adapt to varying environments during the evolution. Here, we devised a strategy called sequence co-evolutionary analysis to control the efficiency of enzyme reactions (SCANEER), which scans the evolution of protein sequences and direct mutation strategy to improve enzyme activity. We hypothesized that amino acid pairs for various enzyme activity were encoded in the evolutionary history of protein sequences, whereas loss-of-function mutations were avoided since those are depleted during the evolution. SCANEER successfully predicted the enzyme activities of beta-lactamase and aminoglycoside 3′-phosphotransferase. SCANEER was further experimentally validated to control the activities of three different enzymes of great interest in chemical production: cis-aconitate decarboxylase, α-ketoglutaric semialdehyde dehydrogenase, and inositol oxygenase. Activity-enhancing mutations that improve substrate-binding affinity or turnover rate were found at sites distal from known active sites or ligand-binding pockets. We provide SCANEER to control desired enzyme activity through a user-friendly webserver.

Engineering of the Substrate Pocket of α-ketoglutaric Semialdehyde Dehydrogenase for Improving the Activity toward 3-hydroxypropanal

Engineering of the Substrate Pocket of α-ketoglutaric Semialdehyde Dehydrogenase for Improving the Activity toward 3-hydroxypropanal

Biochemical and Structural Characterization of l-2-Keto-3-deoxyarabinonate Dehydratase: A Unique Catalytic Mechanism in the Class I Aldolase Protein Superfamily.

l-2-Keto-3-deoxyarabinonate (l-KDA) dehydratase (AraD) catalyzes the hydration of l-KDA to α-ketoglutaric semialdehyde in the nonphosphorylative l-arabinose pathway from bacteria and belongs to the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein superfamily. All members of this superfamily, including several aldolases for l-KDA, share a common catalytic mechanism of retro-aldol fission, in which a lysine residue forms a Schiff base with the carbonyl C2 atom of the substrate, followed by proton abstraction of the substrate by a tyrosine residue as the base catalyst. Only AraD possesses a glutamine residue instead of this active site tyrosine, suggesting its involvement in catalysis. We herein determined the crystal structures of AraD from the nitrogen-fixing bacterium Azospirillum brasilense and AraD in complex with β-hydroxypyruvate and 2-oxobutyrate, two substrate analogues, at resolutions of 1.9, 1.6, and 2.2 Å, respectively. In both of the complexed structures, the ε-nitrogen of the conserved Lys171 was covalently linked to the carbonyl C2 atom of the ligand, which was consistent with the Schiff base intermediate form, similar to other DHDPS/NAL members. A site-directed mutagenic study revealed that Glu173 and Glu200 played important roles as base catalysts, whereas Gln143 was not absolutely essential. The abstraction of one of the C3 protons of the substrate (but not the O4 hydroxyl) by Glu173 was similar to that by the (conserved) tyrosine residues in the two DHDPS/NAL members that produce α-ketoglutaric semialdehyde (d-5-keto-4-deoxygalactarate dehydratase and Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase), indicating that these enzymes evolved convergently despite similarities in the overall reaction.

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria

Alone at last! – Heterologous expression of a single gene is sufficient for establishing the five-step Weimberg pathway in Corynebacterium glutamicum

Corynebacterium glutamicum can grow on d-xylose as sole carbon and energy source via the five-step Weimberg pathway when the pentacistronic xylXABCD operon from Caulobacter crescentus is heterologously expressed. More recently, it could be demonstrated that the C. glutamicum wild type accumulates the Weimberg pathway intermediate d-xylonate when cultivated in the presence of d-xylose. Reason for this is the activity of the endogenous dehydrogenase IolG, which can also oxidize d-xylose. This raised the question whether additional endogenous enzymes in C. glutamicum contribute to the catabolization of d-xylose via the Weimberg pathway. In this study, analysis of the C. glutamicum genome in combination with systematic reduction of the heterologous xylXABCD operon revealed that the hitherto unknown and endogenous dehydrogenase KsaD (Cg0535) can also oxidize α-ketoglutarate semialdehyde to the tricarboxylic acid cycle intermediate α-ketoglutarate, the final enzymatic step of the Weimberg pathway. Furthermore, heterologous expression of either xylX or xylD, encoding for the two dehydratases of the Weimberg pathway in C. crescentus, is sufficient for enabling C. glutamicum to grow on d-xylose as sole carbon and energy source. Finally, several variants for the carbon-efficient microbial production of α-ketoglutarate from d-xylose were constructed. In comparison to cultivation solely on d-glucose, the best strain accumulated up to 1.5-fold more α-ketoglutarate in d-xylose/d-glucose mixtures.

Investigating a Novel Metabolic Pathway for Bacteria to Utilize L‐Ascorbate as its Carbon Source

L‐Ascorbate (Vitamin C) is an abundant yet under‐researched carbon source present in the environment. L‐Ascorbate is a well‐known antioxidant that has significant effects of promoting tissue repair, strengthening bones, and boosting the immune system in mammals. Ralstonia eutropha H16 (Cupriavidus necator) is a saprotrophic soil bacterium that is commonly known for its ability to metabolize a great variety and number of substrates for growth and production of polyhydroxybutyrate (bioplastics). Ralstonia eutropha (ReH16) growth with L‐ascorbate was observed, but the pathway by which ReH16 metabolizes L‐ascorbate is unknown. Transcripts were quantified using RNAseq from cells grown with L‐ascorbate or other carbon sources. Putative pathway genes were identified as genes with transcripts upregulated greater than 50‐fold during L‐ascorbate growth. Transformation, conjugation, homologous recombination, and PCR analysis were used to generate and confirm targeted deletions of two pathway genes [h16_B0212 and h16_B1217]. Mutants of both genes were unable to grow with L‐ascorbate as their sole carbon source. Collaborators have characterized both enzymes. H16_B0212 is an aldehyde dehydrogenase that catalyzes oxidation of the δ‐carbon of α‐ketoglutarate semialdehyde. H16_B1217 is an enzyme that catalyzes the formation of a β‐lactone ring and transfer of a carboxyl group. Characterizing the L‐ascorbate metabolic pathway in Ralsontia eutropha will provide insight into a third, yet unknown pathway for L‐ascorbate metabolism.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sulfolobus acidocaldarius Transports Pentoses via a Carbohydrate Uptake Transporter 2 (CUT2)-Type ABC Transporter and Metabolizes Them through the Aldolase-Independent Weimberg Pathway.

Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources, such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus, only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius MW001 grown on peptides alone and peptides in the presence of d-xylose allowed for the identification of the ABC transporter for d-xylose and l-arabinose transport and the gaining of deeper insights into pentose catabolism under the respective growth conditions. The d-xylose/l-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the carbohydrate uptake transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea Single-gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for d-xylose and l-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and d-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD [also known as KDAD]; Saci_1939) was no longer able to catabolize d-xylose or l-arabinose, suggesting the absence of the aldolase-dependent branch in S. acidocaldariusIMPORTANCE Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications, as their optimal growth conditions resemble conditions used in certain biotechnologies such as industrial plant waste degradation. Because of its high genome stability, Sulfolobus acidocaldarius is especially suited as a platform organism for such applications. For use in (ligno)cellulose degradation, it was important to understand pentose uptake and metabolism in S. acidocaldarius This study revealed that only the aldolase-independent Weimberg pathway is required for growth of S. acidocaldarius MW001 on d-xylose and l-arabinose. Moreover, S. acidocaldarius employs a CUT2 ABC transporter for pentose uptake, which is more similar to bacterial than to archaeal ABC transporters. The identification of pentose-inducible promoters will expedite the metabolic engineering of S. acidocaldarius for its development into a platform organism for (ligno)cellulose degradation.

Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD+, for enhancing the production of 3-hydroxypropionic acid

3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD+ regeneration. We engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD+, were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower Km values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD+, less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM).

Structure and function of a decarboxylating Agrobacterium tumefaciens keto-deoxy-d-galactarate dehydratase.

Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce α-ketoglutaric semialdehyde (α-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 Å, 1.5 Å, and 2.1 Å resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.

Evolution of enzymatic activities in the enolase superfamily: galactarate dehydratase III from Agrobacterium tumefaciens C58.

The genome of Agrobacterium tumefaciens C58 encodes 12 members of the enolase superfamily (ENS), eight of which are members of the mandelate racemase (MR) subgroup and, therefore, likely to be acid sugar dehydratases. Using a library of 77 acid sugars for high-throughput screening, one protein (UniProt entry A9CG74; locus tag Atu4196) showed activity with both m-galactarate and d-galacturonate. Two families of galactarate dehydratases had been discovered previously in the ENS, GalrD/TalrD [Yew, W. S., et al. (2007) Biochemistry46, 9564–9577] and GalrD-II [Rakus, J. F., et al. (2009) Biochemistry48, 11546–11558]; these have different active site acid/base catalysis and have no activity with d-galacturonate. A9CG74 dehydrates m-galactarate to form 2-keto-3-deoxy-galactarate but does not dehydrate d-galacturonate as expected. Instead, when A9CG74 is incubated with d-galacturonate, 3-deoxy-d-xylo-hexarate or 3-deoxy-d-lyxo-hexarate is formed. In this reaction, instead of abstracting the C5 proton α to the carboxylate group, the expected reaction for a member of the ENS, the enzyme apparently abstracts the proton α to the aldehyde group to form 3-deoxy-d-threo-hexulosuronate that undergoes a 1,2-hydride shift similar to the benzylic acid rearrangement to form the observed product. A. tumefaciens C58 does not utilize m-galactarate as a carbon source under the conditions tested in this study, although it does utilize d-galacturonate, which is a likely precursor to m-galactarate. The gene encoding A9CG74 and several genome proximal genes were upregulated with d-galacturonate as the carbon source. One of these, a member of the dihydrodipicolinate synthase superfamily, catalyzes the dehydration and subsequent decarboxylation of 2-keto-3-deoxy-d-galactarate to α-ketoglutarate semialdehyde, thereby providing a pathway for the conversion of m-galactarate to α-ketoglutarate semialdehyde.

Discovery of a novel L-lyxonate degradation pathway in Pseudomonas aeruginosa PAO1.

The l-lyxonate dehydratase (LyxD) in vitro enzymatic activity and in vivo metabolic function were assigned to members of an isofunctional family within the mandelate racemase (MR) subgroup of the enolase superfamily. This study combined in vitro and in vivo data to confirm that the dehydration of l-lyxonate is the biological role of the members of this family. In vitro kinetic experiments revealed catalytic efficiencies of ∼104 M–1 s–1 as previously observed for members of other families in the MR subgroup. Growth studies revealed that l-lyxonate is a carbon source for Pseudomonas aeruginosa PAO1; transcriptomics using qRT-PCR established that the gene encoding LyxD as well as several other conserved proximal genes were upregulated in cells grown on l-lyxonate. The proximal genes were shown to be involved in a pathway for the degradation of l-lyxonate, in which the first step is dehydration by LyxD followed by dehydration of the 2-keto-3-deoxy-l-lyxonate product by 2-keto-3-deoxy-l-lyxonate dehydratase to yield α-ketoglutarate semialdehyde. In the final step, α-ketoglutarate semialdehyde is oxidized by a dehydrogenase to α-ketoglutarate, an intermediate in the citric acid cycle. An X-ray structure for the LyxD from Labrenzia aggregata IAM 12614 with Mg2+ in the active site was determined that confirmed the expectation based on sequence alignments that LyxDs possess a conserved catalytic His-Asp dyad at the end of seventh and sixth β-strands of the (β/α)7β-barrel domain as well as a conserved KxR motif at the end of second β-strand; substitutions for His 316 or Arg 179 inactivated the enzyme. This is the first example of both the LyxD function in the enolase superfamily and a pathway for the catabolism of l-lyxonate.

Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-D-galactarate (KDG) dehydratase from Agrobacterium tumefaciens.

D-galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-D-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of D-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-L-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications.

Unraveling the function of paralogs of the aldehyde dehydrogenase super family from Sulfolobus solfataricus

Aldehyde dehydrogenases (ALDHs) have been well established in all three domains of life and were shown to play essential roles, e.g., in intermediary metabolism and detoxification. In the genome of Sulfolobus solfataricus, five paralogs of the aldehyde dehydrogenases superfamily were identified, however, so far only the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) and α-ketoglutaric semialdehyde dehydrogenase (α-KGSADH) have been characterized. Detailed biochemical analyses of the remaining three ALDHs revealed the presence of two succinic semialdehyde dehydrogenase (SSADH) isoenzymes catalyzing the NAD(P)(+)-dependent oxidation of succinic semialdehyde. Whereas SSO1629 (SSADH-I) is specific for NAD(+), SSO1842 (SSADH-II) exhibits dual cosubstrate specificity (NAD(P)(+)). Physiological significant activity for both SSO-SSADHs was only detected with succinic semialdehyde and α-ketoglutarate semialdehyde. Bioinformatic reconstructions suggest a major function of both enzymes in γ-aminobutyrate, polyamine as well as nitrogen metabolism and they might additionally also function in pentose metabolism. Phylogenetic studies indicated a close relationship of SSO-SSALDHs to GAPNs and also a convergent evolution with the SSADHs from E. coli. Furthermore, for SSO1218, methylmalonate semialdehyde dehydrogenase (MSDH) activity was demonstrated. The enzyme catalyzes the NAD(+)- and CoA-dependent oxidation of methylmalonate semialdehyde, malonate semialdehyde as well as propionaldehyde (PA). For MSDH, a major function in the degradation of branched chain amino acids is proposed which is supported by the high sequence homology with characterized MSDHs from bacteria. This is the first report of MSDH as well as SSADH isoenzymes in Archaea.

Control of hydroxyproline catabolism in Sinorhizobium meliloti

Hydroxyproline (Hyp) in decaying organic matter is a rich source of carbon and nitrogen for microorganisms. A bacterial pathway for Hyp catabolism is known; however, genes and function relationships are not established. In the pathway, trans-4-hydroxy-L-proline (4-L-Hyp) is epimerized to cis-4-hydroxy-D-proline (4-D-Hyp), and then, in three enzymatic reactions, the D-isomer is converted via Δ-pyrroline-4-hydroxy-2-carboxylate (HPC) and α-ketoglutarate semialdehyde (KGSA) to α-ketoglutarate (KG). Here a transcriptional analysis of cells growing on 4-L-Hyp, and the regulation and functions of genes from a Hyp catabolism locus of the legume endosymbiont Sinorhizobium meliloti are reported. Fourteen hydroxyproline catabolism genes (hyp), in five transcripts hypR, hypD, hypH, hypST and hypMNPQO(RE)XYZ, were negatively regulated by hypR. hypRE was shown to encode 4-hydroxyproline 2-epimerase and a hypRE mutant grew with 4-D-Hyp but not 4-L-Hyp. hypO, hypD and hypH are predicted to encode 4-D-Hyp oxidase, HPC deaminase and α-KGSA dehydrogenase respectively. The functions for hypS, hypT, hypX, hypY and hypZ remain to be determined. The data suggest 4-Hyp is converted to the tricarboxylic acid cycle intermediate α-ketoglutarate via the pathway established biochemically for Pseudomonas. This report describes the first molecular characterization of a Hyp catabolism locus.

Establishment of Oxidative d -Xylose Metabolism in Pseudomonas putida S12

The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g D-xylose(-1)) and a maximum growth rate of 0.21 h(-1). Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on D-xylose. Only the xylD gene, encoding D-xylonate dehydratase, proved to be essential for establishing an oxidative D-xylose catabolic pathway in P. putida S12. The growth performance on D-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-D-xylonate dehydratase and alpha-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative D-xylose utilization. Gcd activity not only contributes to D-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on D-xylose.

α-Ketoglutaric Semialdehyde Dehydrogenase of Pseudomonas: PROPERTIES OF THE SEPARATELY INDUCED ISOENZYMES

Abstract In Pseudomonas putida, two induced catabolic pathways, from hydroxyproline and from d-glucarate, converge at a common step, dehydrogenation of α-ketoglutaric semialdehyde to α-ketoglutarate. The dehydrogenases respectively induced by hydroxyproline or glucarate were both purified to homogeneity and compared. In contrast to an earlier, more limited comparison, which revealed no reliable difference between them, detailed electrophoretic, structural, immunological, and genetic studies now show them to be distinct proteins, although closely similar in most kinetic properties studied, as well as in molecular weight, subunit status, and amino acid composition. An unexpected finding was the presence of enzymatic activity for the preceding step in the hydroxyproline pathway (Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase) associated with both the glucarate-induced and hydroxyproline-induced dehydrogenases. In each enzyme, the finding of a 4- to 5-fold lower number of separable tryptic peptides compared with the number expected from subunit size and amino acid composition suggests possible sequence redundancy but needs further investigation.

Catabolism of D-gluaric acid to alpha-ketoglutarate in Bacillus megaterium.

Crude cell-free extracts of d-glucarate-grown cells of Bacillus megaterium converted d-glucarate to alpha-keto-beta-deoxy-d-glucarate (KDG). Charcoal-treated cell-free extracts or partially purified enzyme preparations converted KDG to an intermediate which was isolated and identified as 2,5-diketoadipate (DKA). This compound was synthesized, and the cell-free extracts of d-glucarate grown cells were found to catalyze the reduction of nicotinamide adenine dinucleotide (NAD) in its presence. In the absence of NAD, the same enzyme preparation catalyzed the decarboxylation of the DKA to alpha-ketoglutarate semialdehyde (KGS), whereas in the presence of NAD the KGS was subsequently oxidized to alpha-ketoglutarate by alpha-ketoglutarate semialdehyde dehydrogenase. Since galactarate-grown B. megaterium contains a galactarate dehydrase forming KDG, the complete pathway for the metabolism of d-glucarate or galactarate to alpha-ketoglutarate and CO(2) is now known in a gram-positive bacterium.

α-Ketoglutaric Semialdehyde Dehydrogenase of Pseudomonas: PROPERTIES OF THE PURIFIED ENZYME INDUCED BY HYDROXYPROLINE AND OF THE GLUCARATE-INDUCED AND CONSTITUTIVE ENZYMES

Abstract Hydroxyproline-induced α-ketoglutaric semialdehyde dehydrogenase has been purified from a pseudomonas strain and characterized with respect to substrate specificity and other properties. After a variety of growth conditions, pseudomonas extracts also contain a constitutive dehydrogenase, which differs from the induced enzyme in sedimentation and electrophoretic behavior. Although the constitutive enzyme resembles the induced form in many kinetic respects, it is relatively more active with glutaric semialdehyde as a substrate and is tentatively assigned a role in lysine metabolism. Growth on d-glucarate induces a dehydrogenase which, in all kinetic and physical respects examined, appears identical with that induced by growth on hydroxyproline.

Formation of α-Ketoglutaric Semialdehyde from l-2-Keto-3-deoxyarabonic Acid and Isolation of l-2-Keto-3-deoxyarabonate Dehydratase from Pseudomonas saccharophila

Abstract α-Ketoglutaric semialdehyde has been identified as an intermediate in the conversion of l-2-keto-3-deoxyarabonate to α-ketoglutarate by cell-free extracts of Pseudomonas saccharophila. α-Ketoglutarate semialdehyde arises by the intramolecular dismutation of l-2-keto-3-deoxyarabonate. The enzyme, l-2-keto-3-deoxyarabonate dehydratase, which catalyzes the conversion of l-2-keto-3-deoxyarabonate to α-ketoglutarate semialdehyde, has been purified and crystallized. The enzyme is homogeneous on starch gel electrophoresis and shows a single asymmetrical peak in the ultracentrifuge. No cofactor has been observed.

Constitutive and inducible isozymes of α-ketoglutaric semialdehyde dehydrogenase in Pseudomonas

Constitutive and inducible isozymes of α-ketoglutaric semialdehyde dehydrogenase in Pseudomonas