The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58.

Haloalkane dehalogenases catalyze the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly-isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide-stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen-bonding patterns within the active sites of the wild-type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7-fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K(0.5) for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.

Plant ALDH10 family: identifying critical residues for substrate specificity and trapping a thiohemiacetal intermediate.

Plant ALDH10 family members are aminoaldehyde dehydrogenases (AMADHs), which oxidize ω-aminoaldehydes to the corresponding acids. They have been linked to polyamine catabolism, osmoprotection, secondary metabolism (fragrance), and carnitine biosynthesis. Plants commonly contain two AMADH isoenzymes. We previously studied the substrate specificity of two AMADH isoforms from peas (PsAMADHs). Here, two isoenzymes from tomato (Solanum lycopersicum), SlAMADHs, and three AMADHs from maize (Zea mays), ZmAMADHs, were kinetically investigated to obtain further clues to the catalytic mechanism and the substrate specificity. We also solved the high resolution crystal structures of SlAMADH1 and ZmAMADH1a because these enzymes stand out from the others regarding their activity. From the structural and kinetic analysis, we can state that five residues at positions 163, 288, 289, 444, and 454 (PsAMADHs numbering) can, directly or not, significantly modulate AMADH substrate specificity. In the SlAMADH1 structure, a PEG aldehyde derived from the precipitant forms a thiohemiacetal intermediate, never observed so far. Its absence in the SlAMADH1-E260A structure suggests that Glu-260 can activate the catalytic cysteine as a nucleophile. We show that the five AMADHs studied here are capable of oxidizing 3-dimethylsulfoniopropionaldehyde to the cryo- and osmoprotectant 3-dimethylsulfoniopropionate. For the first time, we also show that 3-acetamidopropionaldehyde, the third aminoaldehyde besides 3-aminopropionaldehyde and 4-aminobutyraldehyde, is generally oxidized by AMADHs, meaning that these enzymes are unique in metabolizing and detoxifying aldehyde products of polyamine degradation to nontoxic amino acids. Finally, gene expression profiles in maize indicate that AMADHs might be important for controlling ω-aminoaldehyde levels during early stages of the seed development.

Sphingobium czechense sp. nov., isolated from a hexachlorocyclohexane dump site.

A yellow-pigmented bacterial strain, designated LL01(T), was isolated from hexachlorocyclohexane (HCH)-contaminated soil at Spolana Neratovice, a former Czech producer of lindane. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL01(T) occupied a distinct phylogenetic position in the Sphingobium cluster, showing highest similarity to Sphingobium rhizovicinum CC-FH12-1(T) (98.5 %). The DNA G+C content of strain LL01(T) was 66.1 mol%. The predominant respiratory pigment was ubiquinone Q-10. The polar lipid profile of strain LL01(T) also corresponded to those reported for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, sphingoglycolipids), supporting its identification as a member of the genus Sphingobium. Spermidine was the major polyamine observed. The results obtained from DNA-DNA hybridization and biochemical and physiological tests clearly distinguished strain LL01(T) from closely related species of the genus Sphingobium. Therefore, strain LL01(T) represents a novel species of the genus Sphingobium, for which the name Sphingobium czechense sp. nov. is proposed (type strain LL01(T) = CCM 7979(T) = DSM 25410(T)).

Novosphingobium barchaimii sp. nov., isolated from hexachlorocyclohexane-contaminated soil.

A yellow-pigmented bacterial strain, designated LL02(T), was isolated from hexachlorocyclohexane-contaminated soil from Spolana Neratovice, a former Czech producer of lindane. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL02(T) occupied a distinct phylogenetic position in the genus Novosphingobium and showed the highest sequence similarity with Novosphingobium resinovorum NCIMB 8767(T) (98.59 %). DNA-DNA relatedness between strain LL02(T) and its closest phylogenetic neighbours was <70 %, which indicated that strain LL02(T) represented a novel species of the genus Novosphingobium. The DNA G+C content of strain LL02(T) was 67.72±0 mol%. The major respiratory quinone was ubiquinone Q-10. The polar lipid profile of the isolate corresponded to those reported for other members of the genus Novosphingobium (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipids), thus supporting its classification in the genus. Spermidine was the major polyamine. The major fatty acids were summed feature 3 (consisting of C(16 : 1)ω7c and/or C(16 : 1)ω6c; 40.13 %), summed feature 8 (consisting of C(18 : 1)ω7c and/or C(18 : 1)ω6c; 31.09 %) and C(14 : 0) 2-OH (23.16 %). The results obtained from DNA-DNA hybridization and biochemical and physiological tests clearly distinguished the isolate from its closest phylogenetic neighbours. Thus, strain LL02(T) represents a novel species of the genus Novosphingobium, for which the name Novosphingobium barchaimii sp. nov. is proposed. The type strain is LL02(T) ( = CCM 7980(T)  = DSM 25411(T)).

Sphingobium baderi sp. nov., isolated from a hexachlorocyclohexane dump site.

A Gram-stain-negative, rod-shaped and white-coloured bacterial strain, designated LL03(T), was isolated from hexachlorocyclohexane-contaminated soil at Spolana Neratovice, Czech Republic, where lindane was formerly produced. Strain LL03(T) was found to be a degrader of α-, γ- and δ-isomers of hexachlorocyclohexane, although no significant degradation activity was observed for the ß-isomer. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL03(T) occupied a distinct phylogenetic position in the Sphingobium cluster, showing the highest similarity with Sphingobium wenxiniae JZ-1(T) (99.2 %). The DNA G+C content of strain LL03(T) was 67.0 mol%. DNA-DNA relatedness values of strain LL03(T) with its close phylogenetic neighbours were below the threshold level of 70 %, supporting its identification as a representative of a novel species of the genus Sphingobium. The predominant respiratory quinone was ubiquinone Q-10. The polar lipid profile of strain LL03(T) also corresponded to those reported for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipid), supporting its identification as a member of the genus Sphingobium. Spermidine was identified as the major polyamine. The predominant fatty acids were 16 : 0, summed feature 3 (16 : 1ω7c and/or 16 : 1ω6c), summed feature 8 (18 : 1ω7c and/or 18 : 1ω6c) and 14 : 0 2-OH. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty acids C(18 : 1)ω7c, C(16 : 0) and C(14 : 0) 2-OH and the G+C content of the genomic DNA supported the affiliation of the strain to the genus Sphingobium. The results obtained after DNA-DNA hybridization, biochemical and physiological tests clearly distinguished it from closely related species of the genus Sphingobium. Therefore, strain LL03(T) represents a novel species of the genus Sphingobium for which the name Sphingobium baderi LL03(T) sp. nov. is proposed; the type strain is LL03(T) ( = CCM 7981(T) = DSM 25433(T)).