2-Keto-3-deoxy-6-phosphogalactonate Aldolase as a Catalyst for Stereocontrolled Carbon−Carbon Bond Formation

Henderson, Darla P.; Cotterill, Ian C.; Shelton, Michael C.; Toone, Eric J.

doi:10.1021/jo9718814

Cited by 32 publications

(18 citation statements)

References 43 publications

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“…In many cases the Delay-Doudoroff pathway exists in these organisms as an equivalent route for the catabolism of galactose (47). The enzymes of this pathway are often inducibly expressed and include an alternative KDPGal aldolase with the opposite facial selectivity (48). In the case of S. solfataricus the lack of facial selectivity in the aldolase-catalyzed reaction is unusual and permits the same enzyme to be used for the cleavage of KDG and KDGal, both yielding glyceraldehyde and pyruvate.…”

Section: Discussionmentioning

confidence: 99%

Metabolic Pathway Promiscuity in the Archaeon Sulfolobus solfataricus Revealed by Studies on Glucose Dehydrogenase and 2-Keto-3-deoxygluconate Aldolase

Lamble

Heyer

Bull

et al. 2003

Journal of Biological Chemistry

137

206

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The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.The hyperthermophilic Archaeon Sulfolobus solfataricus grows optimally at 80 -85°C and pH 2-4, utilizing a wide range of carbon and energy sources (1). It has become one of the most comprehensively researched model organisms of archaeal metabolism and bioenergetics (2). Central metabolism in this organism involves a modified Entner-Doudoroff pathway (3), production of acetyl-CoA by pyruvate:ferredoxin oxidoreductase (4), and the citric acid cycle coupled to oxidative phosphorylation (5). The modified Entner-Doudoroff pathway is a nonphosphorylative variant of the classic pathway and proceeds with no net production of ATP (Fig. 1). An analogous pathway has also been detected in the thermoacidophilic Archaea Sulfolobus acidocaldarius (6), Thermoplasma acidophilum (7), and Thermoproteus tenax (8), as well as strains of Aspergillus fungi (9, 10).The first reaction of the non-phosphorylative Entner-Doudoroff pathway involves the NAD(P)-dependent oxidation of glucose to gluconate, catalyzed by glucose dehydrogenase. Gluconate is then dehydrated by gluconate dehydratase to 2-keto-3-deoxygluconate (KDG), 1 which undergoes an aldolate cleavage to pyruvate and glyceraldehyde, catalyzed by KDG aldolase. Glyceraldehyde dehydrogenase then oxidizes glyceraldehyde to glycerate, which is phosphorylated by glycerate kinase to give 2-phosphoglycerate. A second molecule of pyruvate is produced from this by the actions of enolase and pyruvate kinase.Glucose dehydrogenase has previously been purified to homogenei...

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Section: Discussionmentioning

confidence: 99%

Metabolic Pathway Promiscuity in the Archaeon Sulfolobus solfataricus Revealed by Studies on Glucose Dehydrogenase and 2-Keto-3-deoxygluconate Aldolase

Lamble

Heyer

Bull

et al. 2003

Journal of Biological Chemistry

137

206

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“…GlcA and GalA aldolases (Scheme 8.1) are specific for cleavage of 2-keto-3-deoxy-6-phospho-d-gluconate (7) and d-galactonate (8), respectively; they are produced by bacteria for the degradation of 6-phosphogluconate or galactonate to give pyruvate 1 and d-glyceraldehyde-3-phosphate (6) [16]. GlcA enzymes from different microorganisms have tolerance for polar short-chain aldehydes, albeit at rather low reaction rates, whereas simple aliphatic or aromatic aldehydes are not converted [16,[49][50][51][52][53]. GlcA mutants were identified with up to 2000-fold improved selectivity for unnatural substrates and 40-fold improved catalytic efficiency [54].…”

Section: Related Pyruvate Aldolases/2-oxobutyrate Aldolasesmentioning

confidence: 99%

“…GalA acts on 2-keto-3-deoxy-6-phospho-d-galactonate (8, Scheme 8.1) and is less abundant [52]. GalA from E. coli was recently cloned, expressed, and characterized [57].…”

Section: Related Pyruvate Aldolases/2-oxobutyrate Aldolasesmentioning

confidence: 99%

Enzyme‐Catalyzed Aldol Additions

Clapés¹,

Joglar²

2013

Modern Methods in Stereoselective Aldol Reactions

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“…2–4 We have focused our efforts on evolving Escherichia coli (EC) 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase and a closely related isoform from the thermophilic bacteria Thermatoga maritima (TM). These aldolases preferentially catalyze si -stereo facial aldol addition reactions between pyruvate and a range of aldehydic electrophiles 5–7 (Figure 1). The types of structures afforded by the enzyme have proven useful in the synthesis of Nikkomycins.…”

Section: Introductionmentioning

confidence: 99%

Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate

Cheriyan

Walters

Kang

et al. 2011

Bioorganic & Medicinal Chemistry

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The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a non-functionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the KM value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of kcat/KM up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced “uniform binding” of the substrates, which is the first step in the evolution of novel catalytic activity.

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2-Keto-3-deoxy-6-phosphogalactonate Aldolase as a Catalyst for Stereocontrolled Carbon−Carbon Bond Formation

Cited by 32 publications

References 43 publications

Metabolic Pathway Promiscuity in the Archaeon Sulfolobus solfataricus Revealed by Studies on Glucose Dehydrogenase and 2-Keto-3-deoxygluconate Aldolase

Metabolic Pathway Promiscuity in the Archaeon Sulfolobus solfataricus Revealed by Studies on Glucose Dehydrogenase and 2-Keto-3-deoxygluconate Aldolase

Enzyme‐Catalyzed Aldol Additions

Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate

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