Crystal structure of uronate dehydrogenase from Agrobacterium tumefaciens

Tarja Parkkinen, Harry Boer, Janne Jänis, Martina Andberg, Merja Penttilä, Anu Koivula, Juha Rouvinen (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

24 Citations (Scopus)

Abstract

Uronate dehydrogenase from Agrobacterium tumefaciens (AtUdh) belongs to the short-chain dehydrogenase/reductase superfamily and catalyzes the oxidation of D-galacturonic acid and D-glucuronic acid with NAD + as a cofactor. We have determined the crystal structures of an apo-form of AtUdh, a ternary form in complex with NADH and product (substrate-soaked structure), and an inactive Y136A mutant in complex with NAD +. The crystal structures suggest AtUdh to be a homohexamer, which has also been observed to be the major form in solution. The monomer contains a Rossmann fold, essential for nucleotide binding and a common feature of the short-chain dehydrogenase/reductase family enzymes. The ternary complex structure reveals a product, D-galactaro-1,5-lactone, which is bound above the nicotinamide ring. This product rearranges in solution to D-galactaro-1,4-lactone as verified by mass spectrometry analysis, which agrees with our previous NMR study. The crystal structure of the mutant with the catalytic residue Tyr-136 substituted with alanine shows changes in the position of Ile-74 and Ser-75. This probably altered the binding of the nicotinamide end of NAD +, which was not visible in the electron density map. The structures presented provide novel insights into cofactor and substrate binding and the reaction mechanism of AtUdh. This information can be applied to the design of efficient microbial conversion of D-galacturonic acid-based waste materials.

Original languageEnglish
Pages (from-to)27294-27300
JournalJournal of Biological Chemistry
Volume286
Issue number31
DOIs
Publication statusPublished - 5 Aug 2011
MoE publication typeA1 Journal article-refereed

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uronate dehydrogenase
Agrobacterium tumefaciens
NAD
Oxidoreductases
Crystal structure
Niacinamide
Lactones
Glucuronic Acid
Substrates
Alanine
Carrier concentration
Mass spectrometry
Mass Spectrometry
Nucleotides
Monomers
Nuclear magnetic resonance
Electrons
Oxidation
Enzymes

Cite this

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title = "Crystal structure of uronate dehydrogenase from Agrobacterium tumefaciens",
abstract = "Uronate dehydrogenase from Agrobacterium tumefaciens (AtUdh) belongs to the short-chain dehydrogenase/reductase superfamily and catalyzes the oxidation of D-galacturonic acid and D-glucuronic acid with NAD + as a cofactor. We have determined the crystal structures of an apo-form of AtUdh, a ternary form in complex with NADH and product (substrate-soaked structure), and an inactive Y136A mutant in complex with NAD +. The crystal structures suggest AtUdh to be a homohexamer, which has also been observed to be the major form in solution. The monomer contains a Rossmann fold, essential for nucleotide binding and a common feature of the short-chain dehydrogenase/reductase family enzymes. The ternary complex structure reveals a product, D-galactaro-1,5-lactone, which is bound above the nicotinamide ring. This product rearranges in solution to D-galactaro-1,4-lactone as verified by mass spectrometry analysis, which agrees with our previous NMR study. The crystal structure of the mutant with the catalytic residue Tyr-136 substituted with alanine shows changes in the position of Ile-74 and Ser-75. This probably altered the binding of the nicotinamide end of NAD +, which was not visible in the electron density map. The structures presented provide novel insights into cofactor and substrate binding and the reaction mechanism of AtUdh. This information can be applied to the design of efficient microbial conversion of D-galacturonic acid-based waste materials.",
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Crystal structure of uronate dehydrogenase from Agrobacterium tumefaciens. / Parkkinen, Tarja; Boer, Harry; Jänis, Janne; Andberg, Martina; Penttilä, Merja; Koivula, Anu; Rouvinen, Juha (Corresponding Author).

In: Journal of Biological Chemistry, Vol. 286, No. 31, 05.08.2011, p. 27294-27300.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Crystal structure of uronate dehydrogenase from Agrobacterium tumefaciens

AU - Parkkinen, Tarja

AU - Boer, Harry

AU - Jänis, Janne

AU - Andberg, Martina

AU - Penttilä, Merja

AU - Koivula, Anu

AU - Rouvinen, Juha

N1 - CA2: TK404 CA2: TK400 SDA: BIC ISI: BIOCHEMISTRY & MOLECULAR BIOLOGY

PY - 2011/8/5

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AB - Uronate dehydrogenase from Agrobacterium tumefaciens (AtUdh) belongs to the short-chain dehydrogenase/reductase superfamily and catalyzes the oxidation of D-galacturonic acid and D-glucuronic acid with NAD + as a cofactor. We have determined the crystal structures of an apo-form of AtUdh, a ternary form in complex with NADH and product (substrate-soaked structure), and an inactive Y136A mutant in complex with NAD +. The crystal structures suggest AtUdh to be a homohexamer, which has also been observed to be the major form in solution. The monomer contains a Rossmann fold, essential for nucleotide binding and a common feature of the short-chain dehydrogenase/reductase family enzymes. The ternary complex structure reveals a product, D-galactaro-1,5-lactone, which is bound above the nicotinamide ring. This product rearranges in solution to D-galactaro-1,4-lactone as verified by mass spectrometry analysis, which agrees with our previous NMR study. The crystal structure of the mutant with the catalytic residue Tyr-136 substituted with alanine shows changes in the position of Ile-74 and Ser-75. This probably altered the binding of the nicotinamide end of NAD +, which was not visible in the electron density map. The structures presented provide novel insights into cofactor and substrate binding and the reaction mechanism of AtUdh. This information can be applied to the design of efficient microbial conversion of D-galacturonic acid-based waste materials.

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SP - 27294

EP - 27300

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