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S C Tu

Publications and source records attributed to S C Tu.

At least 37 records · Page 2Linked to original sources
PubMed1996

Gene overexpression, purification, and identification of a desulfurization enzyme from Rhodococcus sp. strain IGTS8 as a sulfide/sulfoxide monooxygenase.

The oxidation of dibenzothiophene to dibenzothiophene sulfone has been linked to the enzyme encoded by the sox/dszC gene from Rhodococcus sp. strain IGTS8 (S. A. Denome, C. Oldfield, L. J. Nash, and K. D. Young, J. Bacteriol. 176:6707-6717, 1994; C. S. Piddington, B. R. Kovacevich, and J. Rambosek, Appl. Environ. Microbiol. 61:468-475, 1995). However, this enzyme has not been characterized, and the type of its catalytic activity remains unclassified. In this work, the sox/dszC gene was overexpressed in Escherichia coli, a procedure for the purification of the expressed enzyme was developed, and the properties of and the reactions catalyzed by the purified enzyme were characterized. This enzyme binds one flavin mononucleotide (Kd, 7 micrometers) or reduced flavin mononucleotide (FMNH2) (Kd < 10(-8) M) per 90,200-Da homodimer, and FMNH2 is an essential cosubstrate for its activity. Patterns of product formation were examined under different FMNH2 availabilities, and results indicate that this enzyme catalyzes a stepwise conversion of dibenzothiophene to the corresponding sulfoxide and subsequently to the sulfone. On the basis of isotope labeling patterns with H2(18)O and 18O2, dibenzothiophene sulfoxide and sulfone obtained their oxygen atom(s) from molecular oxygen rather than water in their formation from dibenzothiophene. The enzyme also utilizes benzyl sulfide and benzyl sulfoxide as substrates. Hence, it is identified as a sulfide/sulfoxide monooxygenase. This monooxygenase is similar to the microsomal flavin-containing monooxygenase but is unique among microbial flavomonooxygenases in its ability to catalyze two consecutive monooxygenation reactions.

Bacterial Proteins↗
PubMed1995

Catalytically active forms of the individual subunits of Vibrio harveyi luciferase and their kinetic and binding properties.

Contradictory findings have recently been reported regarding the (in)abilities of individual subunits of the Vibrio harveyi alpha beta dimeric luciferase to catalyze bioluminescence. We have produced individual alpha and beta subunits separately in Escherichia coli JM109 cells by recombinant DNA techniques. Both subunits were purified to more than 90% homogeneity and found to be catalytically active, with their general catalytic properties and the specific activities similar to those reported earlier (Sinclair, J. F., Waddle, J. J., Waddill, E. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044). Individual subunits were significantly distinct from the native luciferase with respect to inactivations by trypsin and N-ethylmaleimide, and the stability of the flavin 4a-hydroperoxide intermediate. The active species in isolated alpha and beta samples were each the predominant protein species, corresponding to a 42,000 M(r) alpha monomer and a 67,000 M(r) beta dimer, respectively. These findings clearly indicate that the activities of the individual subunits are not due to trace contaminations of the respective counter subunits. The much reduced specific activities of the individual subunits are, in part, a consequence of diminished abilities to oxidize the aldehyde substrate. Kinetic and equilibrium measurements indicate that alpha and beta 2 each contained a reduced flavin site, an aldehyde substrate site, and an aldehyde inhibitor site. The on and off rates of the decanal inhibitor binding were substantially slower than the bindings of decanal and reduced riboflavin 5'-phosphate substrates. These findings are consistent with a scheme that the aldehyde inhibitor blocks the binding of the reduced flavin substrate.

Binding Sites↗
PubMed1994

Probing the Vibrio harveyi luciferase beta subunit functionality and the intersubunit domain by site-directed mutagenesis.

While the critical role of the bacterial luciferase alpha subunit in catalysis has been amply documented, the beta subunit was only known to be involved in thermal stability and substrate binding. Two conserved histidyl residues at position 81 and 82 of the beta subunit of Vibrio harveyi luciferase were each mutated to an alanine, aspartate, or lysine to probe further the beta functionality. These mutations resulted in higher Km values for reduced riboflavin 5'-phosphate, less efficient oxidations of the aldehyde substrate, and decreased light-emitting activities. beta His82 appears to be significantly more critical than beta His81. For the beta His82-mutated luciferases, the maximal light intensities and total light outputs were reduced to 19-4% of that for the wild-type enzyme, and the values of Vmax/Km,flavin were decreased by 2-3 orders of magnitude. The reduced light emission activities for these mutated luciferases can be correlated to lower yields of the flavin 4a-hydroperoxide intermediate, reduced productions of the excited flavin emitter, and/or enhanced quenching of the emitter. The beta subunit and the conserved beta His82 in particular have thus been shown to be critical not only to flavin binding but also to catalytic characteristics of luciferase. The dimeric structure of luciferase is essential to its high catalytic efficiency. To characterize the intersubunit domain, three sets of single/double mutants were constructed, and the additivities of mutational effects were tested to screen for residues that could interact across the subunit interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallography, X-Ray↗
PubMed1994

Crystallization and preliminary crystallographic analysis of NADPH:FMN oxidoreductase from Vibrio harveyi.

Crystals of NADPH:FMN oxidoreductase from Vibrio harveyi have been obtained and characterized by X-ray diffraction. This enzyme plays a role in the generation of light in luminescent bacteria by providing reduced FMN to luciferase. Large, high quality crystals were grown using polyethylene glycol 6000 at pH 7.0. They crystallize in the monoclinic space group P2(1) with cell dimensions a = 51.2 A, b = 85.9 A, c = 58.1 A, beta = 109.3 degrees, and diffract to 1.8 A. We expect two molecules per asymmetric unit. High resolution data sets have been recorded and a search is under way for heavy-atom derivatives.

Crystallization↗
PubMed1994

Mechanism of aldehyde inhibition of Vibrio harveyi luciferase. Identification of two aldehyde sites and relationship between aldehyde and flavin binding.

Vibrio harveyi luciferase is sensitive to aldehyde substrate inhibition, and two kinetic schemes have been previously postulated to account for such an inhibition. One scheme depicts a sequential binding of 2 aldehyde molecules, yielding an active enzyme-aldehyde binary complex and subsequently an inactive enzyme-(aldehyde)2 ternary complex (Holzman, T. F., and Baldwin, T. O. (1983) Biochemistry 22, 2838-2846). This two-aldehyde model was later withdrawn, and recently, a different scheme was proposed, following which the prior binding of one aldehyde to the native luciferase forms an inactive dead-end complex (Abu-Soud, H. M., Clark, A. C., Francisco, W. A., Baldwin, T. O., and Raushel, F. M. (1993) J. Biol. Chem. 268, 7699-7706). In this work, kinetic and equilibrium studies were carried out to elucidate further the mechanism of aldehyde inhibition. Two, presumably independent, aldehyde-binding sites were detected, with a higher affinity site for the aldehyde substrate and a weaker affinity site for the aldehyde inhibitor. Binding to and dissociation from the inhibitor site by decanal were revealed by chemical relaxation analysis to be slow processes. Furthermore, whereas the binding of the decanal substrate enhances the affinity of the reduced riboflavin 5'-phosphate (FMNH2) site, the binding of decanal to the inhibitor site competes against FMNH2 binding, thus resulting in inhibition of luciferase activity. These findings are not compatible with either of the two earlier schemes mentioned above. A new kinetic model is formulated for the mechanism of aldehyde inhibition. Theoretical kinetic behaviors predicted on the basis of this model are in excellent agreement with experimental observations. A particularly reactive cysteine (residue 106) on the alpha subunit has been previously demonstrated to be at or near an aldehyde site (Fried, A., and Tu, S.-C. (1984) J. Biol. Chem. 259, 10754-10759). Evidence is presented to indicate that this residue is at or near the aldehyde inhibitor site. Relative locations of this residue and binding sites for FMNH2, the aldehyde substrate, and the aldehyde inhibitor are proposed.

Aldehydes↗
PubMed1994

Plasma protein C activity is enhanced by arsenic but inhibited by fluorescent humic acid associated with blackfoot disease.

Blackfoot disease is a peripheral vascular disease causally related to the fluorescent humic acid found in the drinking water of endemic areas in Taiwan. We compared the effects of humic acid (HA) purified from the well water of Blackfoot disease endemic areas with the effects of commercial humic acid (Aldrich) as well as trivalent arsenic (As2O3) on protein C activity, which plays an important role in regulation of blood coagulation and fibrinolysis. Humic acid, either purified from drinking water or obtained commercially, dose-dependently inhibited both activated protein C activity and the activation of protein C induced by Protac, a snake venom-derived protein C activator. In contrast to humic acid, arsenic oxide dose-dependently enhanced both activated protein C activity and the Protac-stimulated activation of protein C. In the presence of humic acid the enhancement effect of arsenic oxide was completely abolished, resulting in concentration-dependent inhibition of protein C activity. Therefore, the results of this study indicate that humic acid is a potent protein C inhibitor even in the presence of arsenic, which enhances the protein C activity. Since protein C is a potent anticoagulant and profibrinolytic agent, acquired defects of protein C induced by humic acid might cause a thrombophilic or hypercoagulable state. Whether this is one of the possible mechanisms of humic acid-induced thrombotic disorders in Blackfoot disease needs to be further characterized.

Arsenic↗
PubMed1994

Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme.

NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism.

Amino Acid Sequence↗
PubMed1993

Fluorescent polyene aliphatics as spectroscopic and mechanistic probes for bacterial luciferase: evidence against carbonyl product from aldehyde as the primary excited species.

The fluorescent alpha-parinaric acid (alpha-PAC) and beta-parinaric acid (beta-PAC) were converted to the corresponding aldehydes and alcohols all of which exhibited absorption and fluorescence properties closely resembling those of the parent acids. alpha-PAC and beta-PAC each binds to luciferase in competition with aldehyde. The hydrophobic nature of the aldehyde site was indicated by the enhanced fluorescence quantum yields of the bound alpha-PAC and beta-PAC. These two polyene acids and the beta-parinaryl alcohol were shown to stabilize the luciferase flavin-peroxide intermediate. alpha-Parinaraldehyde (alpha-PAD) and beta-parainaraldehyde (beta-PAD) were active substrates for Vibrio harveyi and Vibrio fischeri luciferases and, for the former enzyme, exhibited Km values similar to and quantum yields about 20-30% as those for decanal and dodecanal. For the V. harveyi luciferase with reduced FMN as a co-substrate, the alpha-PAD- or beta-PAD-initiated luminescence was indistinguishable from the normal emission obtained with octanal (lambda max 495 nm) showing no additional 430-nm component correlatable with emission from excited alpha-PAC or beta-PAC. In reactions using reduced 2-thioFMN for V. harveyi luciferase or reduced FMN for V. fischeri luciferase plus yellow fluorescent protein, the replacement of octanal by beta-PAD again resulted in no additional 430-nm emission. The lack of any emission correlatable with excited alpha-PAC, beta-PAC, or equivalent carbonyl product was not due to the quenching of the polyene moiety by chemical transformation, binding to luciferase, or a 100% energy transfer to the flavin 4a-hydroxide emitter.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehydes↗
PubMed1993

Construction and characterization of hybrid luciferases coded by lux genes from Xenorhabdus luminescens and Vibrio fischeri.

Molecular cloning techniques were employed to obtain hybrid luciferases with their alpha and beta subunits encoded by luxA and luxB genes, respectively, from Xenorhabdus luminescens strain HW or Vibrio fischeri. Although the two wild-type luminous bacteria are phylogenetically diverged, the hybrid luciferase Xf comprising an alpha from X. luminescens HW and a beta from V. fischeri and the hybrid luciferase VI comprising an alpha from V. fischeri and a beta from X. luminescens HW were both functional in bioluminescence. Their general kinetic properties were close to the wild-type enzymes from which the alpha subunit was derived. The X. luminescens HW enzyme is distinct in having a high optimal temperature for in vitro bioluminescence, a high thermal stability and a sensitivity to aldehyde substrate inhibition. Comparisons of the Xf and VI hybrid luciferases with the two wild-type enzymes indicated that these unusual properties of the X. luminescens HW luciferase originated primarily from the alpha subunit.

Base Sequence↗
PubMed1991

Functional consequences of site-directed mutation of conserved histidyl residues of the bacterial luciferase alpha subunit.

The available sequences for the different bacterial luciferases reveal five conserved histidyl residues at positions 44, 45, 82, 224, and 285 of the alpha subunit. Ten variants of Vibrio harveyi luciferase were obtained by selective site-directed mutations of these five histidines. The essentiality of alpha His44 and alpha His45 was indicated by 4-7 orders of magnitude of bioluminescence activity reductions resulting from the substitution of either histidine by alanine (alpha H44A or alpha H45A), aspartate (alpha H44D or alpha H45D), or lysine (alpha H45K). Moreover, alpha H44A and alpha H45A were distinct from the native luciferase in thermal stabilities. Mutations at the other three positions also resulted in activity reductions ranging from a fewfold to 3 orders of magnitude. Despite these widely different bioluminescence light outputs, mutated luciferases exhibited, in nonturnover in vitro assays, light emission decay rates mostly similar to that of the native luciferase using octanal, decanal, or dodecanal as a substrate. This is attributed to a similarity in the catalytic rate constants of the light-emitting pathway for the native and mutated luciferases, but various mutated luciferases suffer in different degrees from competing dark reaction(s). In accord with this interpretation, the bioluminescence activities of mutated luciferases showed a general parallel with the relative stabilities of their 4a-hydroperoxyflavin intermediate species. Furthermore, the drastically reduced bioluminescence activities for luciferases with the alpha His44 or alpha His45 substituted by aspartate, alanine, or lysine were accompanied by little or no activities for consuming the aldehyde substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

EEG spectral analysis and topographic mapping in Wilson's disease.

Computerized EEG spectral analysis and topographic mapping were performed on 14 patients with Wilson's disease (WD) and 10 normal subjects of comparable ages. The predominant EEG changes in WD were diffuse but uneven topographic abnormalities with a decrease in alpha activity, an increase in theta and delta activities, and a low voltage background mainly in the alpha frequency band. Eleven patients (80%) had at least one of the above EEG changes. Furthermore, topographic mapping provided more clearly defined foci of slowing and epileptiform activity. Patients with cerebral white matter involvement, akinetic-rigid syndrome, dystonia, or psychiatric symptoms tended to have more abnormal EEGs. It is concluded that EEG changes in WD are common and the quantitative EEG analysis can increase the likelihood of detecting mild or even subtle EEG abnormalities in individual patients as well as in the patient group.

Adolescent↗
PubMed1991

Cloning and nucleotide sequences of lux genes and characterization of luciferase of Xenorhabdus luminescens from a human wound.

Xenorhabdus luminescens HW is the only known luminous bacterium isolated from a human (wound) source. A recombinant plasmid was constructed that contained the X. luminescens HW luxA and luxB genes, encoding the luciferase alpha and beta subunits, respectively, as well as luxC, luxD, and a portion of luxE. The nucleotide sequences of these lux genes, organized in the order luxCDABE, were determined, and overexpression of the cloned luciferase genes was achieved in Escherichia coli host cells. The cloned luciferase was indistinguishable from the wild-type enzyme in its in vitro bioluminescence kinetic properties. Contrary to an earlier report, our findings indicate that neither the specific activity nor the size of the alpha (362 amino acid residues, Mr 41,389) and beta (324 amino acid residues, Mr 37,112) subunits of the X. luminescens HW luciferase was unusual among known luminous bacterial systems. Significant sequence homologies of the alpha and beta subunits of the X. luminescens HW luciferase with those of other luminous bacteria were observed. However, the X. luminescens HW luciferase was unusual in the high stability of the 4a-hydroperoxyflavin intermediate and its sensitivity to aldehyde substrate inhibition.

Amino Acid Sequence↗
PubMed1990

Elicitation of an oxidase activity in bacterial luciferase by site-directed mutation of a noncatalytic residue.

Flavin-dependent external monooxygenases and oxidases could catalyze the same flavin oxidation reaction involving distinct mechanisms. To gain insights into enzyme structure-function relationship, site-directed mutagenesis was carried out for Vibrio harveyi luciferase, a monooxygenase. The substitution of the alpha subunit cysteine 106 by alanine shows unambiguously that the alphaCys106 is not essential to catalysis. The corresponding substitution by valine resulted in a substantial reduction of the bioluminescence activity correlatable with the induction of a new flavin oxidation activity typical for oxidases. These findings indicate that mutation of a single noncatalytic residue at the active center of a flavoenzyme could transform one enzyme type to another, thus highlighting the subtlety of enzyme active site structure in relation to catalysis and the versatility of enzyme evolution.

Catalysis↗
PubMed1990

Dithionite treatment of flavins: spectral evidence for covalent adduct formation and effect on in vitro bacterial bioluminescence.

Intrigued by the apparent requirement of dithionite for FMN reduction (as opposed to photoreduction or catalytic hydrogenation) in the H2O2-initiated bacterial bioluminescence reaction, we chose 5-ethyl-3-methyllumiflavinium cation I as a model to investigate possible flavin adduct formation by treatment with dithionite or (bi)sulfite. In the range of pH 5-8, the reaction of dithionite with 5-ethyl-3-methyllumiflavinium cation, which is in equilibrium with the 5-ethyl-4a-hydroxy-3-methyl-4a, 5-dihydrolumiflavin pseudobase II (X = OH), is not limited to the formation of flavosemiquinone and dihydroflavin following two one-electron steps. Several parallel and sequential reactions may take place involving the intermediacy of covalent flavin adducts. Addition of (bi)sulfite gave a 4a-sulfiteflavin adduct II (X = SO3-). Consistent with the S2O4(2-) in equilibrium with 2 SO2-. equilibrium, the reaction of dithionite and II (X = OH; SO3-) gave rise to two flavin adducts in competitive nucleophilic displacements: a 4a-sulfoxylate-flavin radical (II, X = SO2.) and a 4a-dithioniteflavin adduct (II, X = S2O4-), respectively. On increasing the (S2O4(2-), SO2.-)/flavin ratio under N2, the formation of the 4a-sulfoxylate-flavin radical became predominant. The II (X = SO2.) so formed was in equilibrium with the flavosemiquinone and bisulfate and can be trapped by reacting with hydroxylamine. In the initial presence of oxygen, II (X = SO2.) was highly reactive toward O2, giving a fast oxidation to II (X = SO3-) and effectively suppressing the formation of the flavosemiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)

Dithionite↗
PubMed1990

Electrochemical superoxidation of flavins: generation of active precursors in luminescent model systems.

Using 3-methyllumiflavin and tetraacetyliriboflavin as examples, we have shown that the socalled "fully oxidized" flavins can be "superoxidized" at an anodic potential of 1.8 to 1.9 V giving flavin radical cation transients which are rapidly transformed in subsequent chemical reactions. An attack by H2O subsequent to the superoxidation of 3-methyllumiflavin provides a route for the formation of 4a-hydroxy-3-methyllumiflavin radical cation, as evident from the subsequent decomposition to the protonated form of the starting flavin. When 3-methyllumiflavin is superoxidized in the presence of a base, a recycling process occurs, allowing superoxidized flavin to be trapped in a slower, competitive conversion. The relatively more stable trapped product is active in reacting with H2O2 to emit chemiluminescence. Electrochemical oxidation of H2O2 in acetonitrile at 1.30 V in the presence of an oxidized flavin results in a direct protonation of the flavin by H+ generated from the electrolysis of H2O2. Minor reactions presumably provide alternative formations of the 4a-hydroperoxy- and 4a-hydroxy-flavin radical cation transients by the direct addition of HOO. and HO. radicals, which also arise in the oxidation of H2O2, to protonated flavin. Under such conditions the superoxidized flavin radical cation is apparently also formed, either directly or by process(es) such as decomposition of the flavin 4a-adduct radical cations. Subsequent reductions of either the superoxidized flavin or the flavin 4a-adduct radical cations lead to an almost steady level of luminescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Flavins↗
PubMed1989

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida.

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.

Cloning, Molecular↗