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Identification in Agrobacterium Tumefaciens of the D-Galacturonic Acid Dehydrogenase Gene

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Identification in Agrobacterium Tumefaciens of the D-Galacturonic Acid Dehydrogenase Gene Appl Microbiol Biotechnol (2010) 86:901–909 DOI 10.1007/s00253-009-2333-9 BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Identification in Agrobacterium tumefaciens of the D-galacturonic acid dehydrogenase gene Harry Boer & Hannu Maaheimo & Anu Koivula & Merja Penttilä & Peter Richard Received: 27 August 2009 /Revised: 26 October 2009 /Accepted: 26 October 2009 /Published online: 17 November 2009 # Springer-Verlag 2009 Abstract There are at least three different pathways for the Keywords meso-galactaric acid . Mucic acid . Lactone . catabolism of D-galacturonate in microorganisms. In the Oxidative pathway. EC 1.1.1.203 oxidative pathway, which was described in some prokary- otic species, D-galacturonate is first oxidised to meso- galactarate (mucate) by a nicotinamide adenine dinucleotide Introduction (NAD)-dependent dehydrogenase (EC 1.1.1.203). In the following steps of the pathway mucate is converted to 2- D-Galacturonate is the primary monomer of pectin and keto-glutarate. The enzyme activities of this catabolic consequently an important carbon source for microorganisms pathway have been described while the corresponding gene living on decaying plant material. It is also of importance in sequences are still unidentified. The D-galacturonate dehy- biotechnology when cheap raw materials such as pectin-rich drogenase was purified from Agrobacterium tumefaciens, materials like citrus peel or sugar beet pulp are exploited. and the mass of its tryptic peptides was determined using Currently, these raw materials are either dumped or used as MALDI-TOF mass spectrometry. This enabled the identifica- cattle feed. It is thus desirable to find other applications for tion of the corresponding gene udh. It codes for a protein with these raw materials which are available in vast amounts. It 267 amino acids having homology to the protein family of has been suggested to ferment D-galacturonic acid to ethanol NAD(P)-binding Rossmann-fold proteins. The open reading using genetically engineered microbes but also other products frame was functionally expressed in Saccharomyces cerevi- can be envisaged (Doran-Peterson et al. 2008). It is thus siae. The N-terminally tagged protein was not compromised important to have a detailed knowledge of the catabolic in its activity and was used after purification for a kinetic pathways and their enzyme properties. Several entirely characterization. The enzyme was specific for NAD and different pathways for the catabolism of D-galacturonate in accepted D-galacturonic acid and D-glucuronic acid as microorganisms have been described (for review see (Richard substrates with similar affinities. NMR analysis showed that and Hilditch 2009)): in water solution the substrate D-galacturonic acid is (1) The isomerase pathway is active in Escherichia coli predominantly in pyranosic form which is converted by the and other prokaryotic microorganisms. In this path- enzyme to 1,4 lactone of galactaric acid. This lactone seems way, D-galacturonic acid is converted to D-tagaturonate stable under intracellular conditions and does not spontane- by an isomerase followed by a conversion through an ously open to the linear meso-galactaric acid. NADH-dependent reductase to D-altronate. In the next step, a dehydratase forms 3-deoxy-D-erythro-hex-2- ulosonate followed by a phosphorylation at C6 by an : : : : ATP-requiring kinase. The reaction product is then split H. Boer H. Maaheimo A. Koivula M. Penttilä by an aldolase to pyruvate and D-glyceraldehyde-3- * P. Richard ( ) phosphate. The enzyme activities have been character- VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT Espoo, Finland ised (Ashwell et al. 1960; Cynkin and Ashwell 1960; e-mail: [email protected] Hickman and Ashwell 1960; Smiley and Ashwell 1960; 902 Appl Microbiol Biotechnol (2010) 86:901–909 Meloche and Wood 1964) and the corresponding genes following step is the α-keto glutarate semialdehyde identified (Mata-Gilsinger and Ritzenthaler 1983). dehydrogenase (EC 1.2.1.26) where the reaction (2) The reductive pathway has been described in the product is α-ketoglutarate (Watanabe et al. 2007; eukaryotic microorganisms, filamentous fungi Hypo- Fig. 1). This route is also sometimes called the α- crea jecorina and Aspergillus nidulans.Inthis ketoglutarate pathway. pathway D-galacturonate is reduced by an NAD(P)H- requiring reductase to L-galactonate and then converted by a dehydratase to 3-deoxy-L-threo-hex-2-ulosonate, None of the gene sequences for this pathway in A. which is in the next step split by an aldolase to L- tumefaciens or P. syringae are known; however, gene glyceraldehyde and pyruvate. L-Glyceraldehyde is sequences for bacterial galactarate dehydratase (Hubbard oxidised to glycerol in an NADPH-requiring reaction. et al. 1998) and α-keto glutaric semialdehyde dehydroge- The enzymes have been characterised and the nase (Watanabe et al. 2007) in other pathways have been corresponding genes identified (Kuorelahti et al. 2005, described. There is a catabolic path for galactaric acid 2006; Liepins et al. 2006;Hilditchetal.2007;Martens- (mucic acid) in E. coli where the first enzyme is a Uzunova 2008). galactarate dehydratase of which the gene sequence is (3) The oxidative pathway has been described in some known (Hubbard et al. 1998). The following steps of the E. bacteria such as Agrobacterium tumefaciens and coli pathway however are differing from the oxidative D- Pseudomonas syringae. In this path, D-galacturonate galacturonate pathway. This pathway is sometimes called is first oxidised to meso-galactarate (mucate) by an the glycerate pathway (Sharma and Blumenthal 1973). A 5- NAD-requiring D-galacturonate dehydrogenase. meso- keto-4-deoxy-galactarate (3-deoxy-2-oxo-L-threo-hexarate) Galactarate is then converted by a dehydratase to from the dehydration reaction is split by an aldolase into 3-deoxy-2-keto-DL-threo-hexarate (2-keto-3-deoxy pyruvate and L-tartronate semialdehyde. L-Tarartronate galactarate). In the following step, this is converted semialdehyde is then reduced to form D-glycerate which is by a 4-deoxy-5-oxoglucarate hydrolase (decarboxylat- subsequently phosphorylated to D-glycerate-3-phosphate. ing; EC 4.2.1.41) to α-keto glutarate semialdehyde. In The E. coli pathway requires a specific galactarate this enzyme reaction, a dehydratation and decarboxyl- dehydratase. If the enzyme would convert mucic acid to ation is completed in a single step (Jeffcoat 1975). The the L- and D-threo form of 3-deoxy-2-keto hexarate, it could Fig. 1 Pathway for D-galacturonate catabolism in A. tumefaciens: D- galactarate; however, it is not known which stereoisomer is formed. In galacturonate is oxidised by an NAD-dependent dehydrogenase to the following step, a dehydratase-decarboxylase is forming 2- galactaro-lactone. The galactaro-lactone hydrolyses spontaneously or ketoglutarate semialdehyde which is in the last step converted to 2- withtheaidofalactonasetomeso-galactarate (mucate). A ketoglutarate by an NAD-dependent dehydrogenase dehydratase is converting the meso-galactarate to 2-keto-3-deoxy- Appl Microbiol Biotechnol (2010) 86:901–909 903 lead to metabolites with inverse configuration which cannot be were identified using a dehydrogenase-specific zymogram metabolised. In the A. tumefaciens pathway on the other hand, staining in essence as described previously (Richard et al. the product of the pathway, 2-oxo-glutarate, has no chiral 2001). The gel was stained in a solution containing 10 mM centres, accordingly, a more unspecific dehydratase would not Tris-HCl, pH 7.5, 0.5 mM NAD, 4 mM D-galacturonic be harmful. Indeed the galactarate dehydratase of A. tumefa- acid, 0.25 mM nitroblue tetrazolium and 0.06 mM phena- ciens does not seem to be very specific. It was suggested to be zine methosulfate. The only band that appeared in the active with meso-galactaric acid and D-glucaric acid, and from staining was cut out and eluted by overnight incubation in D-glucaric acid the D-erythro and L-threo form of 3-deoxy-2- 2 ml of 100 mM Tris-HCl, pH 9.0, 0.1% SDS. It was then oxo-hexarate was formed (Chang and Feingold 1970). E. coli concentrated to about 80 μl by centrifugation in a Centricon on the other hand has specific enzymes for the dehydratation tube (Amicon) and separated on a sodium dodecyl sulfate of meso-galactaric acid or D-glucaric acid. polyacrylamide gel electrophoresis (SDS-PAGE; 12% A D-galacturonate dehydrogenase (EC 1.1.1.203) has acrylamide). A 32 kDa enzyme observed in the SDS- been described in A. tumefaciens (Chang and Feingold PAGE gel was in-gel digested with trypsin, and the peptides 1969) and in P. syringae (Bateman et al. 1970; Wagner and were extracted essentially according to the method of Hollmann 1976b). The enzyme from these organisms had Rosenfeld et al. (1992).The samples were desalted using a been partially purified and characterised; however, a gene C-18 matrix (Eppendorf Perfect Pure C-18 Tip). The sequence for the corresponding enzyme acitivity has not saturated matrix solution was prepared by dissolving been identified. The enzyme is specific for NAD as a recrystallized α-cyano-4-hydroxycinnamic acid (Bruker cofactor but is not specific for the substrate. It can oxidise Daltonics, Bremen, Germany) in a 50% acetonitrile in D-galacturonate and D-glucuronate to meso-galactarate 0.1% TFA solution. Equal volumes of purified peptide (mucate) and D-glucarate (saccharate), respectively. We sample or calibration standard (peptide calibration mixture present here the purification of the enzyme induced on D- II, Bruker Daltonics) were mixed with the saturated matrix galacturonic acid and the cloning of the corresponding gene solution. One microlitre of this matrix/sample mixture was from A. tumefaciens. The heterologously expressed enzyme applied onto the target (Target plate ground steel T F, was characterised in more detail using 1H nuclear magnetic Bruker Daltonics) and let to dry at room temperature. The resonance (NMR). peptide masses were then determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectometry using a Bruker Autoflex II mass Materials and methods spectrometer.
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