Activation of <i>Sulfolobus solfataricus</i> Alcohol Dehydrogenase by Modification of Cysteine Residue 38 with Iodoacetic Acid

Raia, Carlo A.; Caruso, Carla; Marino, M.; Vespa, Nunzia; Rossi, Mosè

doi:10.1021/bi9502093

Cited by 15 publications

(17 citation statements)

References 31 publications

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“…Similar optimum pHs were observed when benzyl alcohol was used instead of 1-hexanol (data not shown). Although a similar pH profile is observed with A. pernix ADH (10), the optimum pH (pH 10.5) for oxidation catalyzed by ST0053-ADH is more alkaline than that for the S. solfataricus ADH (around pH 9) (22).…”

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confidence: 65%

Sulfolobus tokodaiiST0053 Produces a Novel Thermostable, NAD-Dependent Medium-Chain Alcohol Dehydrogenase

Yanai

Doi

Ohshima

2009

Appl Environ Microbiol

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An alcohol dehydrogenase (ADH) gene, ST0053, from Sulfolobus tokodaii was expressed in Escherichia coli. The purified recombinant enzyme was an NAD-dependent medium-chain ADH with high thermostability and tolerance of a wide range of pHs. This is the first step in creating an experimental functionality library of 10 genes annotated as ADHs in the S. tokodaii genome.NAD(P)-dependent alcohol dehydrogenases (ADHs; EC 1.1.1.1 and EC 1.1.1.2) catalyze the reversible oxidation of alcohols to their corresponding aldehydes or ketones. Many kinds of ADHs have been identified in a variety of microorganisms and are utilized for the production and detection of alcohols, aldehydes, and ketones (1, 11). Particularly useful are ADHs from thermophiles and hyperthermophiles, which are often more stable than their counterparts from mesophiles and psychrophiles (6, 7). These thermostable enzymes provide us with long-term functionality and novel reaction systems, such as the enzymatic vapor reaction under high temperature. Ten different genes have been annotated as NAD(P)-dependent ADHs in the genome of the hyperthermophilic and acidophilic archaeon Sulfolobus tokodaii strain 7 (13). We have been interested in compiling the structural and functional characteristics of their products into a library. Such a library would provide much useful information that could facilitate their application. We here describe the expression of ST0053, one of the 10 ADH genes in the S. tokodaii genome, and the subsequent purification and characteristics of its product.Cloning and expression of ST0053. A hybrid DNA plasmid, pET11a/ST0053, whose ST0053 open reading frame was amplified by PCR and inserted into the NdeI-BamHI site of pET11a (Novagen), was provided by Seiki Kuramitsu (Department of Biology, Graduate School of Science, Osaka University, Osaka, Japan). The plasmid was transformed into E. coli strain Rosetta(DE3), after which the transformants were cultured for 16 h in 10 ml of Luria-Bertani medium containing ampicillin (50 g ml Ϫ1 ) at 37°C with shaking at 120 rpm. This culture medium was then added to 1 liter of Luria-Bertani medium containing the same amount of antibiotic in a 2-liter Erlenmeyer flask and incubated at 37°C with shaking at 220 rpm. Once the optical density at 660 nm reached 0.5, expression was induced by the addition of isopropyl-␤-D-thiogalactopyranoside (IPTG; 1 mM final concentration) and cultivation was continued at 37°C for an additional 4 h. The culture was harvested by centrifugation at 10,000 ϫ g for 10 min at 4°C, the cells were washed with 0.85% NaCl and pelleted, and then the cell pellet was stored at Ϫ80°C.Purification. To purify ADH, the stored cells (wet weight, about 2.55 g) were suspended in 10.2 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 0.01% 2-mercaptoethanol (2-ME) (buffer A) and then disrupted by ultrasonication on ice (Tomy Ultrasonic Disruptor UD-201 set at an output level of 2 and a 50% duty cycle; three sonications for 3 min each with 5-min intervals). The resultant homogenate was centrifuged a...

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supporting

confidence: 65%

Sulfolobus tokodaiiST0053 Produces a Novel Thermostable, NAD-Dependent Medium-Chain Alcohol Dehydrogenase

Yanai

Doi

Ohshima

2009

Appl Environ Microbiol

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“…Recently, two types of ADH were isolated from hyperthermophilic organisms and characterized. One was a group I, Zn-containing ADH from Sulfolobus solfataricus (50,52), while the other type has been obtained from three Thermococcus species ( Table 7). The complete sequence of one of the Thermococcus enzymes, i.e., that from T. zilligii (formerly Thermococcus strain AN1 [31,54]) is available and shows similarities with sequences of the group III Fe-dependent ADHs (31).…”

Section: Discussionmentioning

confidence: 99%

An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Adams

1999

J Bacteriol

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Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100°C by the fermentation of peptides and carbohydrates to produce acetate, CO2, and H2, together with minor amounts of ethanol. The organism also generates H2S in the presence of elemental sulfur (S0). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S0. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 ± 1,000, appears to be a homohexamer, and contains iron (∼1.0 g-atom/subunit) and zinc (∼1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent Km s (at 80°C) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.

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“…Indeed, some MDRs fulfil all these requirements (e.g. broad ADH subfamily [133,136,137], or animal ADH subfamily [15,138]).…”

Section: Is There a Mdr Ancestral Activity?mentioning

confidence: 99%

Diversity, taxonomy and evolution of medium‐chain dehydrogenase/reductase superfamily

Riveros‐Rosas

Julián‐Sánchez

Villalobos‐Molina

et al. 2003

European Journal of Biochemistry

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A comprehensive, structural and functional, in silico analysis of the medium-chain dehydrogenase/reductase (MDR) superfamily, including 583 proteins, was carried out by use of extensive database mining and the BLASTP program in an iterative manner to identify all known members of the superfamily. Based on phylogenetic, sequence, and functional similarities, the protein members of the MDR superfamily were classified into three different taxonomic categories: (a) subfamilies, consisting of a closed group containing a set of ideally orthologous proteins that perform the same function; (b) families, each comprising a cluster of monophyletic subfamilies that possess significant sequence identity among them and might share or not common substrates or mechanisms of reaction; and (c) macrofamilies, each comprising a cluster of monophyletic protein families with protein members from the three domains of life, which includes at least one subfamily member that displays activity related to a very ancient metabolic pathway. In this context, a superfamily is a group of homologous protein families (and/or macrofamilies) with monophyletic origin that shares at least a barely detectable sequence similarity, but showing the same 3D fold.The MDR superfamily encloses three macrofamilies, with eight families and 49 subfamilies. These subfamilies exhibit great functional diversity including noncatalytic members with different subcellular, phylogenetic, and species distributions. This results from constant enzymogenesis and proteinogenesis within each kingdom, and highlights the huge plasticity that MDR superfamily members possess. Thus, through evolution a great number of taxa-specific new functions were acquired by MDRs. The generation of new functions fulfilled by proteins, can be considered as the essence of protein evolution. The mechanisms of protein evolution inside MDR are not constrained to conserve substrate specificity and/or chemistry of catalysis. In consequence, MDR functional diversity is more complex than sequence diversity.MDR is a very ancient protein superfamily that existed in the last universal common ancestor. It had at least two (and probably three) different ancestral activities related to formaldehyde metabolism and alcoholic fermentation. Eukaryotic members of this superfamily are more related to bacterial than to archaeal members; horizontal gene transfer among the domains of life appears to be a rare event in modern organisms.Keywords: protein taxonomy; protein evolution; mediumchain alcohol dehydrogenase; enoyl reductase; formaldehyde dehydrogenase.Correspondence to H. Riveros-Rosas, Depto. Bioquı´mica, Fac. Medicina, UNAM, Apdo. Postal 70-159, Cd. Universitaria, Me´xico, 04510, D.F., Me´xico. Fax: + 52 55 5616 2419, Tel.: + 52 55 5622 0829, E-mail: hriveros@servidor.unam.mx Abbreviations: AADH, allyl alcohol dehydrogenase; ACR, acyl-CoA reductase; ADH, alcohol dehydrogenase; AL, alginate lyase; ARP, auxinregulated protein; AST, membrane traffic protein; BCHC, 2-desacetyl-2-hydroxyethyl bacteriochlorophyll...

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Activation of Sulfolobus solfataricus Alcohol Dehydrogenase by Modification of Cysteine Residue 38 with Iodoacetic Acid

Cited by 15 publications

References 31 publications

Sulfolobus tokodaiiST0053 Produces a Novel Thermostable, NAD-Dependent Medium-Chain Alcohol Dehydrogenase

Sulfolobus tokodaiiST0053 Produces a Novel Thermostable, NAD-Dependent Medium-Chain Alcohol Dehydrogenase

An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Diversity, taxonomy and evolution of medium‐chain dehydrogenase/reductase superfamily

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