Biochemical characterization of broad-specificity enzymes using multivariate experimental design and a colorimetric microplate assay: characterization of the haloalkane dehalogenase mutants

Marvanová, Soňa; Nagata, Yasunobu; Wimmerová, Michaela; Sýkorová, Jana; Hynková, Kamila; Damborský, Jiřı́

doi:10.1016/s0167-7012(00)00250-5

Cited by 24 publications

(15 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…HLD activity was tested in a colorimetric assay to detect the production of protons. 27,28 Both DmmA short and DmmA long were active in this assay, however DmmA short exhibited tenfold higher activity than the longer form of the protein ( Table I), suggesting that the N-terminus may interfere with catalysis. Steady-state kinetic constants were determined for DmmA short (Table II), which revealed that these parameters are comparable to other HLDs.…”

Section: Hld Activitymentioning

confidence: 95%

Structure and activity of DmmA, a marine haloalkane dehalogenase

Gehret¹,

Gu²,

Geders³

et al. 2012

Protein Science

View full text Add to dashboard Cite

DmmA is a haloalkane dehalogenase (HLD) identified and characterized from the metagenomic DNA of a marine microbial consortium. Dehalogenase activity was detected with 1,3-dibromopropane as substrate, with steady-state kinetic parameters typical of HLDs (K m 5 0.24 6 0.05 mM, k cat 5 2.4 6 0.1 s 21). The 2.2-Å crystal structure of DmmA revealed a fold and active site similar to other HLDs, but with a substantially larger active site binding pocket, suggestive of an ability to act on bulky substrates. This enhanced cavity was shown to accept a range of linear and cyclic substrates, suggesting that DmmA will contribute to the expanding industrial applications of HLDs.

show abstract

Section: Hld Activitymentioning

confidence: 95%

Structure and activity of DmmA, a marine haloalkane dehalogenase

Gehret¹,

Gu²,

Geders³

et al. 2012

Protein Science

View full text Add to dashboard Cite

show abstract

“…The effect of ionic strength on activity was tested in Tris-H 2 SO 4 buffer (pH 7.5), and the highest activity was observed in 50 mM buffer (data not shown). The substrate specificity of DhmA was tested with 34 different halogenated compounds selected for testing by a statistical experimental design (21). Halide ions formed by hydrolytic dehalogenation of the substrates by DhmA were detected colorimetrically.…”

Section: Vol 68 2002mentioning

confidence: 99%

Cloning and Expression of the Haloalkane Dehalogenase GenedhmAfromMycobacterium aviumN85 and Preliminary Characterization of DhmA

Jesenská¹,

Bartoš

Czerneková

et al. 2002

Appl Environ Microbiol

View full text Add to dashboard Cite

Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G؉C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.Haloalkane dehalogenases catalyze hydrolytic cleavage of carbon-halogen bonds in halogenated aliphatic compounds, leading to the formation of primary alcohols, halide ions, and protons. These enzymes are potentially useful for cleaning up contaminated subsurfaces (32) and for processing by-products of chemical syntheses (33). Haloalkane dehalogenases can serve as a model system for studies of the evolution and distribution of degradation enzymes in the environment since many of these enzymes have already been isolated from different bacterial species originating from geographically distinct areas (26). Haloalkane dehalogenases have primarily been isolated from bacteria colonizing environments contaminated by halogenated substances (12,16,22,(27)(28)(29)(30)40). Only recently have the hydrolytic dehalogenation activities of several species of the genus Mycobacterium isolated from clinical material been reported (14). Motivation for the search for haloalkane dehalogenases in clinical samples of mycobacteria came from the identification of dehalogenase-like genes in the genome of Mycobacterium tuberculosis...

show abstract

“…The activities of purified mycobacterial dehalogenases were determined by a microtiter plate colorimetric assay using the reagents of Iwasaki (14) described previously (26). The amount of 0.2 mg of pure DmbA per ml in the glycine buffer (pH 8.5) was incubated with the halogenated substrate at a final concentration 10 mM.…”

mentioning

confidence: 99%

Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

Jesenská

Pavlová

Strouhal

et al. 2005

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45°C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47°C and DmbB is 57°C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.Haloalkane dehalogenases catalyze detoxification reactions and act on a broad range of halogenated aliphatic compounds (15). Haloalkane dehalogenases have primarily been isolated from bacterial strains living in soil contaminated with halogenated aliphatic compounds (16,20,30,37,38,41,42,45). The enzymes are involved in biochemical pathways enabling bacteria to utilize halogenated compounds, i.e., 1,2-dichloroethane, 1-chloroalkanes, ␥-hexachlorocyclohexane, 1,2-dibromoethane, and 1,3-dichloropropene as the source of carbon and energy.Database searches revealed the presence of at least two open reading frames, rv2579 and rv2296, with sequences highly similar to haloalkane dehalogenases in the genome of the pathogenic bacterium Mycobacterium tuberculosis H37Rv (4). Dehalogenating activity was consequently confirmed in M. tuberculosis H37Rv (19). The same study demonstrated dehalogenase activity in several saprophytic mycobacterial species. The first haloalkane dehalogenase originating from a mycobacterial strain was cloned from Mycobacterium sp. strain GP1 (37). This haloalkane dehalogenase, designated DhaA f , is involved in the biochemical pathway for biodegradation of 1,2-dibromoethane. Genome sequence analysis has shown that two other representatives (fast-growing M. smegmatis MC2 155 and slowgrowing M. avium subsp. paratuberculosis K10), possess open reading frames aal17946, map0345c, and map2057 coding for putative haloalkane dehalogenases....

show abstract

Biochemical characterization of broad-specificity enzymes using multivariate experimental design and a colorimetric microplate assay: characterization of the haloalkane dehalogenase mutants

Cited by 24 publications

References 14 publications

Structure and activity of DmmA, a marine haloalkane dehalogenase

Structure and activity of DmmA, a marine haloalkane dehalogenase

Cloning and Expression of the Haloalkane Dehalogenase GenedhmAfromMycobacterium aviumN85 and Preliminary Characterization of DhmA

Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

Contact Info

Product

Resources

About