Oxidation of Nicotinic Acid by a Bacillus Species: Purification and Properties of Nicotinic Acid and 6-Hydroxynicotinic Acid Hydroxylases

The enzymes of a Bacillus species that hydroxylate nicotinic acid to 6-hydroxynicotinic acid and 6-hydroxynicotinic acid to 2,6-dihydroxynicotinic acid were purified and characterized. The purified enzymes contained approximately two molecules of flavine and eight molecules of iron per molecule of enzyme. The enzymes were large (molecular weight, 400,000 to 450,000) and appeared to consist of subunits.     

Oxidation of Nicotinic Acid by a Bacillus Species: Source of Oxygen Atoms for the Hydroxylation of Nicotinic Acid and 6-Hydroxynicotinic Acid

Three types of evidence are presented to show that the enzymes that hydroxylate nicotinic acid to 2,6-dihydroxynicotinic acid use water as a source of oxygen atoms. (18)O is incorporated into the products from H(2) (18)O. Molecular oxygen acts as a terminal electron acceptor, one-half molecule being consumed per molecule of hydroxyl groups incorporated. An external electron acceptor is required for activity in purified preparations.     

Manganese Oxidation by Spores and Spore Coats of a Marine Bacillus Species

Bacillus sp. strain SG-1 is a marine bacterial species isolated from a near-shore manganese sediment sample. Its mature dormant spores promote the oxidation of Mn²⁺ to MnO₂. By quantifying the amounts of immobilized and oxidized manganese, it was established that bound manganese was almost instantaneously oxidized. When the final oxidation of manganese by the spores was partly inhibited by NaN₃ or anaerobiosis, an equivalent decrease in manganese immobilization was observed. After formation of a certain amount of MnO₂ by the spores, the oxidation rate decreased. A maximal encrustment was observed after which no further oxidation occurred. The oxidizing activity could be recovered by reduction of the MnO₂ with hydroxylamine. Once the spores were encrusted, they could bind significant amounts of manganese, even when no oxidation occurred. Purified spore coat preparations oxidized manganese at the same rate as intact spores. During the oxidation of manganese in spore coat preparations, molecular oxygen was consumed and protons were liberated. The data indicate that a spore coat component promoted the oxidation of Mn²⁺ in a biologically catalyzed process, after adsorption of the ion to incipiently formed MnO₂. Eventually, when large amounts of MnO₂ were allowed to accumulate, the active sites were masked and further oxidation was prevented.     

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.Microbial utilization and degradation of alkanes was discovered almost a century ago (27). Since then, several enzyme families capable of hydroxylating alkanes to alkanols, the first step in alkane degradation, have been identified and categorized based on their preferred substrates (30). The soluble and particulate methane monooxygenases (sMMO and pMMO) and the related propane monooxygenase and butane monooxygenase (BMO) are specialized on gaseous small-chain alkanes (C₁ to C₄), while medium-chain (C₅ to C₁₆) alkane hydroxylation seems to be the domain of the CYP153 and AlkB enzyme families.Conversion of C₁ to C₄ alkanes to alkanols is of particular interest for producing liquid fuels or chemical precursors from natural gas. The MMO-like enzymes that catalyze this reaction in nature, however, exhibit limited stability or poor heterologous expression (30) and have not been suitable for use in a recombinant host that can be engineered to optimize substrate or cofactor delivery. Alkane monooxygenases often cometabolize a wider range of alkanes than those which support growth (12). We wished to determine whether it is possible to engineer a medium-chain alkane monooxygenase to hydroxylate small alkanes, thereby circumventing difficulties associated with engineering MMO-like enzymes as well as investigating the fundamental question of whether enzymes unrelated to MMO can support growth on small alkanes.The most intensively studied medium-chain alkane hydroxylases are the AlkB enzymes (2, 20, 29), especially AlkB from Pseudomonas putida GPo1 (13, 28, 32, 35). While most members of the AlkB family act on C₁₀ or longer alkanes, some accept alkanes as small as C₅ (30). A recent study (12) indicated that AlkB from P. putida GPo1 may also be involved in propane and butane assimilation. AlkB selectively oxidizes at the terminal carbon to produce the 1-alkanols. No systematic protein engineering studies have been conducted on this di-iron integral membrane enzyme, although selection and site-directed mutagenesis efforts identified one amino acid residue that sterically determines long-chain alkane degradation (35).The most recent addition to the known biological alkane-hydroxylating repertoire is the CYP153 family of heme-containing cytochrome P450 monooxygenases. Although their activity was detected as early as 1981 (1), the first CYP153 was characterized only in 2001 (16). Additional CYP153 enzymes were identified and studied more recently (9, 10, 31). These soluble class II-type three-component P450 enzymes and the AlkB enzymes are the main actors in medium-chain-length alkane hydroxylation by the cultivated bacteria analyzed to date (31). CYP153 monooxygenases have been the subject of biochemical studies (9, 16, 19), and their substrate range has been explored (10, 14). Known substrates include C₅ to C₁₁ alkanes. The best-characterized member, CYP153A6, hydroxylates its preferred substrate octane predominantly (>95%) at the terminal position (9).Recent studies have shown that high activities on small alkanes can be obtained by engineering bacterial P450 enzymes such as P450cam (CYP101; camphor hydroxylase) and P450 BM3 (CYP102A; a fatty acid hydroxylase) (8, 36). The resulting enzymes, however, hydroxylate propane and higher alkanes primarily at the more energetically favorable subterminal positions; highly selective terminal hydroxylation is difficult to achieve by engineering a subterminal hydroxylase (22). We wished to determine whether a small-alkane terminal hydroxylase could be obtained instead by directed evolution of a longer-chain alkane hydroxylase that exhibits this desirable regioselectivity. For this study, we chose to engineer AlkB from P. putida GPo1 and CYP153A6 from Mycobacterium sp. strain HXN-1500 (9, 33) to enhance activity on butane. Because terminal alkane hydroxylation is the first step of alkane catabolism in P. putida GPo1, we reasoned that it should be possible to establish an in vivo evolution system that uses growth on small alkanes to select for enzyme variants exhibiting the desired activities.The recombinant host Pseudomonas putida GPo12(pGEc47ΔB) was engineered specifically for complementation studies with terminal alkane hydroxylases and was used previously to characterize members of the AlkB and CYP153 families (26, 31). This strain is a derivative of the natural isolate P. putida GPo1 lacking its endogenous OCT plasmid (octane assimilation) (5) but containing cosmid pGEc47ΔB, which carries all genes comprising the alk machinery necessary for alkane utilization, with the exception of a deleted alkB gene (34). We show that this host can be complemented by a plasmid-encoded library of alkane hydroxylases and that growth of the mixed culture on butane leads to enrichment of novel butane-oxidizing terminal hydroxylases.     

Regulation of Polyglutamic Acid Synthesis by Glutamate in Bacillus licheniformis and Bacillus subtilis

The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular γ-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.     

Predicted Amino Acid Sequence of Bovine Tyrosine Hydroxylase and Its Similarity to Tyrosine Hydroxylases from Other Species

The previously obtained cDNAs coding for bovine tyrosine hydroxylase (TH) mRNA (mRNATH) were further analyzed, and the entire nucleotide sequence was determined. The mRNATH consists of 1,706 nucleotides with an open reading frame for 491 amino acids, which corresponds to a calculated molecular weight of 55,011. The predicted amino acid sequence of bovine TH is compared with that of rat TH and shows a similarity of 66% in the amino terminal (amino acids 1-157) and 91% in the carboxy terminal (amino acids 158-491) region of the TH protein molecule. The carboxy terminal region has been shown to make up the catalytic site of TH and, therefore, is conserved to a greater extent in different species than the amino terminal region, which has been shown to be mainly responsible for the regulation of the catalytic activity of TH. Three of the four serine residues (Ser 8, 19, and 40) that have been shown to be substrates for various protein kinases in rat TH are also present in bovine TH and are located near the amino terminal end of the molecule. The amino acids from position 60 to position 66 of rat TH are not present in bovine TH, resulting in the absence of a predicted hydrophobic region as compared with rat TH. This difference could result in an altered degree of regulation by posttranslational phosphorylation and also association to cell organelle membranes of bovine TH as compared with rat TH.     

Fatty Acid Composition of Lipid Extracts of a Thermophilic Bacillus Species

Studies on the relationship between cell synthesis and energy utilization in Hydrogenomonas eutropha have shown that the amount of oxidative energy required for synthetic reactions depends on the conditions of growth. The energy of hydrogen oxidation was most efficiently used when growth conditions were optimal (continuous culture, cells in exponential growth phase) and when the rate of growth was limited by H(2) or O(2) supply. Under these conditions, 2 to 2.5 atoms of oxygen were consumed by the oxyhydrogen reaction for the concomitant conversion of 1 mole of CO(2) to cell matter. This conversion efficiency, expressed as the O/C energyyield value, was observed with continuous cultures. A less efficient conversion was found with batch cultures. With limiting concentrations of CO(2) the rate of hydrogen oxidation was relatively high, and the O/C value was dependent on the growth rate. With nonlimiting concentrations of CO(2), the rate of hydrogen oxidation was strictly proportional to the rate of CO(2) fixation, and the O/C value was independent of growth rate. This proportionality between the rate of H(2) oxidation and the rate of CO(2) fixation suggested that energy supply regulates the (maximum) rate of growth. From the energy-yield measurements, we concluded that the oxidation of 1 mole of H(2) yields the equivalent of 2 moles of adenosine triphosphate for H. eutropha, and that at least 5 moles of this high-energy phosphate is required for the conversion of 1 mole of CO(2) into cellular constituents.     

Biodegradation of Phenanthrene by a Bacillus Species

A bacterial strain capable of utilizing phenanthrene as sole source of carbon was isolated from soil and identified as a Bacillus sp. The organism also utilized naphthalene, biphenyl, anthracene, and other aromatic compounds as growth substrates. The organism degraded phenanthrene through the intermediate formation of 1-hydroxy-2-naphthoic acid, which was further metabolized via o-phthalate by a protocatechuate pathway, as evidenced by oxygen uptake and enzymatic studies. Received: 1 December 1999 / Accepted: 5 January 2000     

Enzymatic Manganese(II) Oxidation by Metabolically Dormant Spores of Diverse Bacillus Species

Bacterial spores are renowned for their longevity, ubiquity, and resistance to environmental insults, but virtually nothing is known regarding whether these metabolically dormant structures impact their surrounding chemical environments. In the present study, a number of spore-forming bacteria that produce dormant spores which enzymatically oxidize soluble Mn(II) to insoluble Mn(IV) oxides were isolated from coastal marine sediments. The highly charged and reactive surfaces of biogenic metal oxides dramatically influence the oxidation and sorption of both trace metals and organics in the environment. Prior to this study, the only known Mn(II)-oxidizing sporeformer was the marine Bacillus sp. strain SG-1, an extensively studied bacterium in which Mn(II) oxidation is believed to be catalyzed by a multicopper oxidase, MnxG. Phylogenetic analysis based on 16S rRNA and mnxG sequences obtained from 15 different Mn(II)-oxidizing sporeformers (including SG-1) revealed extensive diversity within the genus Bacillus, with organisms falling into several distinct clusters and lineages. In addition, active Mn(II)-oxidizing proteins of various sizes, as observed in sodium dodecyl sulfate-polyacrylamide electrophoresis gels, were recovered from the outer layers of purified dormant spores of the isolates. These are the first active Mn(II)-oxidizing enzymes identified in spores or gram-positive bacteria. Although extremely resistant to denaturation, the activities of these enzymes were inhibited by azide and o-phenanthroline, consistent with the involvement of multicopper oxidases. Overall, these studies suggest that the commonly held view that bacterial spores are merely inactive structures in the environment should be revised.     

Regulation of Brain Nicotinic Receptors by Chronic Agonist Infusion

Several studies have demonstrated that chronic treatment with nicotine elicits an increase in the number of brain nicotinic receptors. To determine whether this effect is elicited by other nicotinic agonists found in tobacco, the effects of chronic infusion with nicotine on brain nicotinic receptors were compared with those after anabasine and lobeline. C57BL/6 mice were infused with saline or equimolar doses (18.5 mumol/kg/h) of nicotine, anabasine, or lobeline for 8 days. Nicotinic receptors, quantified by the binding of [3H]nicotine and [125I]iodo-alpha-bungarotoxin (alpha-[125I]BTX), and muscarinic receptors, quantified by the binding of [3H]quinuclidinyl benzilate ([3H]QNB), were then assayed in eight brain regions. An increase in [3H]nicotine binding was observed in all regions except cerebellum following chronic infusion with nicotine and anabasine, whereas lobeline did not alter the number or affinity of these binding sites. This increase was due to changes in Bmax and not in the affinity of the receptor for the ligand (KD). A slight increase in alpha-[125I]BTX binding was observed in cortex following chronic anabasine infusion. [3H]QNB binding sites were largely unaltered following chronic infusion with any of the nicotinic analogs. The levels of the agonists in the brain were also determined after chronic treatment, and the amounts of lobeline and anabasine were found to be higher than that of nicotine. Thus, the failure of lobeline to elicit changes in nicotine binding is not due to reduced brain concentrations.     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-alpha-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-C]linoleic acid or [5-C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [C]linoleic acid was incorporated into 6-pentyl-alpha-pyrone more rapidly than that from [C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-alpha-pyrone when fungal cultures were incubated with [1-C]linoleic acid. These results suggested that beta-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species.     

Oxidation of Methanol,Formaldehyde and Formate by a Candida Species

1. The oxidation of methanol to carbon dioxide by Candida N–16 grown on methanol was investigated. The presence of enzymes which catalyze the following reaction was found in the cell-free extract of the yeast employed; CH₃OH→HCHO→HCOOH→CO₂. 2. Methanol was oxidized to formaldehyde by an alcohol oxidase. The reaction was as follows; CH₃OH+O₂→HCHO+H₂O₂. The alcohol oxidase was crystallized after purification by ammonium sulfate-precipitation and column chromatography using DEAE-Sephadex A-50. A prosthetic group of the enzyme was proved to be FAD. The enzyme possessed a broad specificity for alcohols such as methanol, ethanol, n-propanol, n-butanol and n-amylalcohol. The enzyme was inducibly formed only by the addition of methanol. 3. The oxidation of formaldehyde to formate was catalyzed by a NAD-linked dehydrogenase dependent on GSH. 4. Formate was oxidized by a NAD-linked dehydrogenase. 5. Catalase was also found in the extract, and methanol was chemically oxidized by the reaction of catalase and hydrogen peroxide which was generated by the alcohol oxidase system. 6. The oxidation pathway from methanol to carbon dioxide was also found in other methanol-utilizing yeasts such as Candida N-17, Saccharomyces H-1 and Torulopsis M-1.     

NAD~+对小麦叶片线粒体甘氨酸氧化和三羧酸代谢的调节

外源NAD~+对小麦叶片线粒体内甘氨酸、苹果酸及α—酮戊二酸氧化都有促进作用。当几种呼吸底物同时存在时,其中甘氨酸的氧化抑制了其他底物的同时氧化,因为催化这两类废物氧化的酶对NAD~+的亲和力和对NADH/NAD~+比值的敏感程度有差异,催化甘氨酸氧化的甘氨酸脱羧酶对线粒体基质内可利用的NAD~+的亲和力分别比苹果酸脱氢酶和α—酮戊二酸脱氢酶的亲和力大约1或2倍。另外,甘氨酸亦可通过保持线粒体基质内高NADH/NAD~+比值来影响三羧酸环的正常代谢。     

Cyanide Formation from Oxidation of Glycine by a Pseudomonas Species

With whole cells of a hydrogen cyanide-producing bacterium strain C, of the genus Pseudomonas, it was found that the oxygen necessary for the oxidation of glycine to cyanide could be replaced by various artificial electron acceptors. The order of reactivity was: oxygen > phenazine methosulphate > methylene blue > 2,6-dichlorophenolindophenol > ferricyanide. Cyanide production was inhibited by pyrrolnitrin, a well-known inhibitor of many flavine enzymes. The molar ratio of added glycine to cyanide produced was found to be 1.09. With whole bacteria the apparent K(m) (glycine) for the cyanide production was found to be 5.0 x 10(-4) M.     

Oxidation of Nitrapyrin to 6-Chloropicolinic Acid by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea

Suspensions of Nitrosomonas europaea catalyzed the oxidation of the commercial nitrification inhibitor nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine]. Rapid oxidation of nitrapyrin (at a concentration of 10 μM) required the concomitant oxidation of ammonia, hydroxylamine, or hydrazine. The turnover rate was highest in the presence of 10 mM ammonia (0.8 nmol of nitrapyrin per min/mg of protein). The product of the reaction was 6-chloropicolinic acid. By the use of ¹⁸O₂, it was shown that one of the oxygens in 6-chloropicolinic acid came from diatomic oxygen and that the other came from water. Approximately 13% of the radioactivity of [2,6-¹⁴C]nitrapyrin was shown to bind to cells. Most (94%) of the latter was bound indiscriminately to membrane proteins. The nitrapyrin bound to membrane proteins may account for the observed inactivation of ammonia oxidation.