Polycyclic aromatic hydrocarbons loads and potential risks in freshwater ecosystem of the Ikpa River Basin, Niger Delta-Nigeria.

In this study, the levels of 16 USEPA-prioritized polycyclic aromatic hydrocarbons (PAHs) were quantified in various environmental matrices in Ikpa River Basin and nearby dumpsites using gas chromatography-mass spectrometry (GC-MS). The levels of the contaminants were further subjected to models to estimate possible sources and potential risks. The results obtained revealed that nearby dumpsites could be the major source of contamination of the Ikpa River Basin. The total sum of PAHs in water and sediment samples gave 926.6 µg/l and 1099.7 µg/kg, respectively. Higher concentrations, 3025.8 µg/kg, 3645.7 µg/kg, and 2457.2 µg/l, were recorded for nearby municipal dumpsite soil, hospital dumpsite soil, and landfill leachates, respectively. Hierarchical cluster analysis (HCA) and PAH molecular diagnostic ratios (MDRs) suggest that PAH loading in the river basin were mostly of pyrogenic origin. The risk assessment indicated that exposure to PAHs through dermal contact with sediments was most significant than oral ingestion of water and children were the most vulnerable group. Non-cancer (toxic) risks due to exposure to PAHs by oral ingestion of water from Ikpa River were within acceptable limits as the calculated hazard quotients (HQs) and hazard indexes (HIs) were below unity, suggesting negligible or no toxic risk. However, toxic risks through dermal contact with sediments reached unacceptable limits as HI values exceeded unity for all sample stations. Estimated cancer risks due to oral ingestion of water reached the USEPA minimum risk level (3.14 × 10(-5)) requiring public notification while risks due to dermal adsorption of PAHs from the sediments reached levels (2.10 × 10(-1)) requiring remediation actions.

Assessment of the Occurrence and Risks of Emerging Organic Pollutants (EOPs) in Ikpa River Basin Freshwater Ecosystem, Niger Delta-Nigeria.

The levels of some emerging organic pollutants (EOPs) including endocrine disrupting compounds, pharmaceuticals and personal care products were quantified in surface water of a freshwater ecosystem, the Ikpa River Basin, Nigeria using liquid chromatography/high resolution tandem mass spectrometry (LC-MS/MS). In addition, leachates and storm water samples collected from nearby dumpsites were also analysed to assess the effect on water quality. Seventeen compounds were detected at the nanogramme-per-litre levels and the ecological risks of selected compounds assessed based on predicted no-effect concentrations derived from comparison of toxicity data recorded for green algae, fish and invertebrate with the maximum measured environmental concentrations, to obtain risk quotients. Some of the compounds showed some level of widespread occurrence or persistence. Also, bisphenol A, chloramphenicol, erythromycin, triclocarban and triclosan were the most important EOPs detected in the study area that may pose detrimental effects to the aquatic organisms based on the outcome of the risk assessment.

Flavonoids biotransformation by bacterial non-heme dioxygenases, biphenyl and naphthalene dioxygenase.

This review details recent progresses in the flavonoid biotransformation by bacterial non-heme dioxygenases, biphenyl dioxygenase (BDO), and naphthalene dioxygenase (NDO), which can initially activate biphenyl and naphthalene with insertion of dioxygen in stereospecfic and regiospecific manners. Flavone, isoflavone, flavanone, and isoflavanol were biotransformed by BDO from Pseudomonas pseudoalcaligenes KF707 and NDO from Pseudomonas sp. strain NCIB9816-4, respectively. In general, BDO showed wide range of substrate spectrum and produced the oxidized products, whereas NDO only metabolized flat two-dimensional substrates of flavone and isoflavone. Furthermore, biotransformation of B-ring skewed substrates, flavanone and isoflavanol, by BDO produced the epoxide products, instead of dihydrodiols. These results support the idea that substrate-driven reactivity alteration of the Fe-oxo active species may occur in the active site of non-heme dioxygenases. The study of flavonoid biotransformation by structurally-well defined BDO and NDO will provide the substrate structure and reactivity relationships and eventually establish the production of non-plant-originated flavonoids by means of microbial biotechnology.

Absolute configuration-dependent epoxide formation from isoflavan-4-ol stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain KF707.

Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2' and C3' on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.

Location of flavone B-ring controls regioselectivity and stereoselectivity of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

Naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 incorporated dioxygen at the C7 and C8 positions on the A-rings of flavone and isoflavone with different stereoselectivity, resulting in the formation of (7S,8S)-dihydroxy-2-phenyl-7,8-dihydro-4H-chromen-4-one (flavone-cis-(7S,8S)-dihydrodiol) and (7R,8R)-dihydroxy-3-phenyl-7,8-dihydro-4H-chromen-4-one (isoflavone-cis-(7R,8R)-dihydrodiol), respectively. In addition, NDO was shown to incorporate dioxygen at the C5 and C6 positions on the A-ring and the C2' and C3' positions on the B-ring of isoflavone, resulting in the production of (5S,6R)-dihydroxy-3-phenyl-5,6-dihydro-4H-chromen-4-one (isoflavone-cis-(5S,6R)-dihydrodiol) and 3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one (isoflavone-cis-(2'R,3'S)-dihydrodiol), respectively. The metabolites were identified by LC/MS, (1)H, and (13)C NMR analyses and TD-SCF calculations combined with CD spectroscopy. In the case of flavone biotransformation, formation of flavone-(7S,8S)-dihydrodiol is likely to be the result of hydrogen bond interactions between the substrate and the active site of the dioxygenase. On the contrary, regioselective dioxygenation of isoflavone was found not to occur, and this may be due to the fact that the same hydrogen bonds that occur in the case of the flavone reaction cannot be established due to steric hindrance caused by the position of the B-ring. It is therefore proposed that the regioselectivity and stereoselectivity of NDO from strain NCIB 9816-4 are controlled by the position of the phenyl ring on flavone molecules.

Time-dependent density functional theory-assisted absolute configuration determination of cis-dihydrodiol metabolite produced from isoflavone by biphenyl dioxygenase.

Escherichia coli cells containing the biphenyl dioxygenase genes bphA1A2A3A4 from Pseudomonas pseudoalcaligenes KF707 were found to biotransform isoflavone and produced a metabolite that was not found in a control experiment. Liquid chromatography/mass spectrometry (LC/MS) and (1)H and (13)C nuclear magnetic resonance (NMR) analyses indicated that biphenyl dioxygenase induced 2',3'-cis-dihydroxylation of the B-ring of isoflavone. In a previous report, the same enzyme showed dioxygenase activity toward flavone, producing flavone 2',3'-cis-dihydrodiol. Due to growing interest in flavone chemistry and the absolute configuration of natural products, time-dependent density functional theory (TD-DFT) calculations were combined with circular dichroism (CD) spectroscopy to determine the absolute configuration of the isoflavone dihydrodiol. By computational methods, the structure of the isoflavone metabolite was determined to be 3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one. This structure was confirmed further by the modified Mosher's method. The same protocol was applied to the flavone metabolite, and the absolute configuration was determined to be 2-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one. After determination of the absolute configurations of the biotransformation products, we suggest the binding mode of these substrate analogs to the enzyme active site.

A key role for vesicles in fungal secondary metabolism.

Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B(1); these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.

Rapid oxidation of ring methyl groups is the primary mechanism of biotransformation of gemfibrozil by the fungus Cunninghamella elegans.

The hypolipidemic agent gemfibrozil (GEM), which has been studied for its metabolism in humans and animals, was investigated to elucidate its primary metabolism by Cunninghamella elegans. The fungus produced ten metabolites (FM1-FM9 and FM6') from the biotransformation of GEM. Based on LC/MS/MS and NMR analyses, a major metabolite, FM7, was identified as 2'-hydroxymethyl GEM. FM6 was considered to be 5'-hydroxymethyl GEM, after comparison of results LC/MS, LC/MS/MS, and UV absorption spectra to FM7. The combined concentration of FM6 and FM7 was found to increase up to 0.83 mM by day 2, and then decreased gradually with incubation time, followed by a noticeable increase in the biotransformation product, FM1, up to 0.86 mM by day 15. NMR analyses confirmed that FM1 was 2',5'-dihydroxymethyl GEM. Further minor oxidations of the aromatic ring and carboxylic acid intermediates were also detected. Based upon these findings, the major fungal metabolic pathway for GEM is likely to occur via production of 2',5'-dihydroxymethyl GEM from 2'-hydroxymethyl GEM. These relatively rapid and diverse biotransformations of GEM by C. elegans suggest that depending upon conditions, it may also follow a similar biodegradation fate when released into the natural environment.

Catalytic mechanism of inulinase from Arthrobacter sp. S37.

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k(cat)/K(m)) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k(cat), but not due to variations in K(m), consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK(a) of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK(a). Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme.

Identification of fungal metabolites of anticonvulsant drug carbamazepine.

Carbamazepine, which has been used in the treatments of epilepsy, is often found in the environment. Although metabolism of carbamazepine by humans and rats has been characterized, the environmental fate of carbamazepine has not been studied. In this study, two model fungi Cunninghamella elegans ATCC 9245 and Umbelopsis ramanniana R-56, which have previously shown diverse metabolic activities, were tested for metabolism of carbamazepine. Both fungi produced three metabolites each (C1-C3 and M1-M3). All six metabolites showed [M + H](+) at m/z 253, suggesting addition of one oxygen to the parent compound. High-performance liquid chromatography and liquid chromatography-mass spectrometric analysis detected 10, 11-dihydro-10, 11-epoxycarbamazepine as a major product (C3 (47%) and M3 (85%)) and 3-hydroxycarbamazepine (C2 (15%) and M2 (7%)) from carbamazepine through mixed mono-oxidation reactions in both fungal strains. C. elegans was confirmed to produce 2-hydroxycarbamazepine (C1 (38%)) while U. ramanniana produced a yet unidentified ring-hydroxylated metabolite (M1 (8%)). The current study suggests that carbamazepine is likely to be subjected to initially diverse mono-oxygenation reactions by fungal metabolisms, resulting in the formation of the corresponding metabolites, which were similarly found in mammalian metabolisms.

Absolute configurations of isoflavan-4-ol stereoisomers.

Isoflavan-4-ol has been synthesized quantitatively from the reduction of isoflavone in the presence of Pd/C and ammonium formate under N(2) atmosphere. Isolation of cis- and trans-isomers was achieved by flash column chromatography and each enantiomer was separated by Sumi-Chiral column chromatography. Absolute configurations of four stereoisomers were determined by circular dichroism spectroscopy.

Reductive dechlorination of methoxychlor and DDT by human intestinal bacterium Eubacterium limosum under anaerobic conditions.

Methoxychlor [1,1,1-trichloro-2,2-bis(p-methoxyphenyl)ethane], a substitute for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), is a compound of environmental concern because of potential long-term health risks related to its endocrine-disrupting and carcinogenic potency. In order to determine the metabolic fate of methoxychlor and DDT in the human intestinal gut, Eubacterium limosum (ATCC 8486), a strict anaerobe isolated from the human intestine that is capable of O-demethylation toward O-methylated isoflavones, was used as a model intestinal microbial organism. Under anaerobic incubation conditions, E. limosum completely transformed methoxychlor and DDT in 16 days. Based on gas chromatography-mass chromatography analyses, the metabolites produced from methoxychlor and DDT by E. limosum were confirmed to be 1,1-dichloro-2,2-bis(p-methoxyphenyl)ethane (methoxydichlor) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), respectively. This study suggests that E. limosum in the human intestinal gut might be a participant in the reductive dechlorination of methoxychlor to the more antiandrogenic active methoxydichlor.

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase.

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.

Metabolic characterization of newly isolated Pseudomonas nitroreducens Jin1 growing on eugenol and isoeugenol.

Newly isolated soil bacterium strain Jin1 was able to grow on both eugenol and isoeugenol each as sole source of carbon and energy. Based on bacterial 16S rDNA analysis, Jin1 belongs to Pseudomonas nitroreducens with a similarity of 98.92% (14/1297). P. nitroreducens Jin1 was found to biotransform eugenol and isoeugenol to vanillin by different pathways. Eugenol was biotransformed to vanillin through coniferyl alcohol and ferulic acid similarly to the pathway shown previously by Pseudomonassp. HR199 and vanillin produced from eugenol was rapidly metabolized to vanillic acid. Contrastively, Pseudomonas nitroreducens Jin1 did not appear to produce metabolic intermediates during the biotransformation of isoeugenol to vanillin which was finally biotransformed to vanillic acid with much slower rate. These results indicate that there seems to be different metabolic regulation systems for the biotransformation of eugenol and isoeugenol by this bacterium. Herein, we report on Pseudomonas nitroreducens Jin1, a novel bacterium that produces vanillin from eugenol and isoeugenol by two different metabolic pathways.

Fungal biodegradation of carbofuran and carbofuran phenol by the fungus Mucor ramannianus: identification of metabolites.

The fungus Mucor ramannianus was tested to elucidate the biological fate of a class of N-methyl carbamate pesticide carbofuran and its hydrolysed form carbofuran phenol. The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column showed that two peaks occurred after incubation of M. ramannianus with which 1 mM carbofuran was combined as a final concentration. In culture of M. ramannianus with 1 mM carbofuran phenol, it produced two other metabolites compared to metabolites of carbofuran. LC/MS analysis suggested that two of the metabolites produced from carbofuran phenol were most likely to be 2-hydoxy-3-(3-methylpropan-2-ol)phenol or 7a-(hydroxymethyl)-2,2-dimethylhexahydro-6H-furo[2,3-b]pyran-6-one and 3-hydroxycarbofuran-7-phenol.

Production of phytoestrogen S-equol from daidzein in mixed culture of two anaerobic bacteria.

An anaerobic incubation mixture of two bacterial strains Eggerthella sp. Julong 732 and Lactobacillus sp. Niu-O16, which have been known to transform dihydrodaidzein to S-equol and daidzein to dihydrodaidzein respectively, produced S-equol from daidzein through dihydrodaidzein. The biotransformation kinetics of daidzein by the mixed cultures showed that the production of S-equol from daidzein was significantly enhanced, as compared to the production of S-equol from dihydrodaidzein by Eggerthella sp. Julong 732 alone. The substrate daidzein in the mixed culture was almost completely converted to S-equol in 24 h of anaerobic incubation. The increased production of S-equol from daidzein by the mixed culture is likely related to the increased bacterial numbers of Eggerthella sp. Julong 732. In the mixture cultures, the growth of Eggerthella sp. Julong 732 was significantly increased while the growth of Lactobacillus sp. Niu-O16 was suppressed as compared to either the single culture of Eggerthella sp. Julong 732 or Lactobacillus sp. Niu-O16. This is the first report in which two metabolic pathways to produce S-equol from daidzein by a mixed culture of bacteria isolated from human and bovine intestinal environments were successfully linked under anaerobic conditions.

Organophosphate pesticide concentrations in coral tissues of Indonesian coastal waters.

In this study we evaluated the persistence of diazinon, chlorpyrifos, profenofos, parathion, malathion and ethion in dead coral tissues of Indonesian coastal waters (Java, Bali, Sulawesi and Komodo). Comparison of the residue levels in coral tissues showed that the highest presence of organophosphate concentrations was detected in a coral sample collected from Java coastal waters. While medium amounts of a contaminant diazinon can still lead to detectable in Bali and Sulawesi coastal waters. Prominent contamination of organophosphate was not found in a sample collected from Komodo. Neither parathion nor malathion were detected in any of the samples. This result implies that the geographical variations of organophosphate compounds are determined by the possible usage of these chemicals around coastal waters at the present or in the past. There is need for further work to identify sources and fate of pesticide contaminants, as well as to improve monitoring of pesticide use.

Characterization of the cytochrome P450 enzymes involved in the metabolism of a new cardioprotective agent KR-33028.

KR-33028 (N-[4-cyano-benzo[b]thiophene-2-carbonyl]guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. This study was performed to characterize the cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-33028. Hydroxylation (5-hydroxy- and 7-hydroxy-KR-33028) is major pathways for the metabolism of KR-33028 in human liver microsomes. Among the nine c-DNA expressed CYP isoforms tested, KR-33028 was 5-hydroxylated by CYP3A4 and 7-hydroxylated by CYP1A2, CYP3A4, and CYP2C19. These findings were supported by the combination of chemical inhibition studies in human liver microsomes and correlation analysis. Furafylline and ketoconazole potently inhibited hydroxylation of KR-33028 in human liver microsomes. Correlation analysis between the known CYP enzyme activities and the rates of the formation of 5-hydroxy- and 7-hydroxy-KR-33028 in the 16 human liver microsomes has showed significant correlations with CYP3A4-mediated midazolam 1'-hydroxylation and CYP1A2-mediated phenacetin O-deethylation, respectively. A 7-hydroxy-KR-33028 formation is also weakly correlated with CYP3A4-mediated midazolam 1'-hydroxylation. The kinetics of the major biotransformation of KR-33028 were studied: CYP3A4 mediated the formation of 5-hydroxy-KR-33028 from KR-33028 with Cl(int)=0.22microl/min/pmol CYP. The intrinsic clearance for 7-hydroxy-KR-33028 formation by CYP1A2, CYP2C19, and CYP3A4 were 0.26, 0.19, and 0.03microl/min/pmol CYP, respectively. Taken together, these results provide evidence that CYP3A4 and CYP1A2 are the major isoforms responsible for the hydroxy metabolites formation from KR-33028.

In vitro metabolism of a new cardioprotective agent, KR-32570, in human liver microsomes.

KR-32570 (5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl)guanidine) is a new reversible Na+/H+ exchanger inhibitor for preventing ischemia-reperfusion injury. This study was performed to identify the metabolic pathway of KR-32570 in human liver microsomes. Human liver microsomal incubation of KR-32570 in the presence of NADPH and UDPGA resulted in the formation of six metabolites, M1-M6. M1 was identified as O-desmethyl-KR-32570, on the basis of liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis with the synthesized authentic standard. M2 and M3 were suggested to be hydroxy-KR-32570 and hydroxy-O-desmethyl-KR-32570, respectively. M1, M2, and M3 were further metabolized to their glucuronide conjugates, M4, M5, and M6, respectively. In addition, the specific P450 isoforms responsible for KR-32570 oxidation to two major metabolites, O-desmethyl-KR-32570 and hydroxy-KR-32570, were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 isoforms. The inhibitory potency of KR-32570 on clinically major P450s was investigated in human liver microsomes. The results show that CYP3A4 contributes to the oxidation of KR-32570 to hydroxy-KR-32570, and CYP1A2 play the predominant role in O-demethylation of KR-32570. KR-32570 was found to inhibit moderately the metabolism of CYP2C8 substrates.

Pyrococcus furiosus alpha-amylase is stabilized by calcium and zinc.

The hyperthermophilic archeon Pyrococcus furiosus produces an extracellular alpha-amylase that belongs to glycosyl hydrolases' family 13. This enzyme is more thermostable than its bacterial and archaeal homologues (e.g., Bacillus licheniformis TAKA-term and Pyrococcus kodakaraensis KOD1 alpha-amylases, respectively) even without adding Ca(2+) ions. Unlike the TAKA-therm amylase that contains no cysteine, the P. furiosus enzyme contains five cysteines (C152, C153, C165, C387, and C430), only four of which (C152, C153, C387, and C430) are conserved in the P. kodakaraensis alpha-amylase. To test the potential function of cysteines in P. furiosus alpha-amylase stability, these five residues were substituted with Ser or Ala-either one-by-one or in sequence-to produce eight mutant enzymes. Mutation C165S dramatically destabilized P. furiosus alpha-amylase. At the same time, the quadruple mutant enzyme C152S/C153S/C387S/C430A (mutant SSCSA) was as thermostable as the wild-type enzyme. Mutant SSCSA and wild-type alpha-amylases were strongly destabilized by dithiothreitol and ethylenediaminetetraacetic acid, suggesting that metal binding can be involved in this enzyme's thermostability. Inductively coupled plasma-atomic emission spectrometry showed the presence of Ca(2+) and Zn(2+) metal ions in P. furiosus alpha-amylase. Although Ca(2+) is known to contribute to alpha-amylase's stability, the absence of two out of the three conserved Ca(2+) ligands in the P. furiosus enzyme suggests that a different set of amino acids is involved in this enzyme's Ca(2+) binding. We also provide evidence suggesting that Cys165 is involved in Zn(2+) binding and that Cys165 is essential for the stability of P. furiosus alpha-amylase at very high temperatures.