Trace metals, enzymes, and biogeochemical cycles

Carbon cycle: carbonic anhydrase

Once taken up and bound within the cells of microorganisms, some trace metals—iron and zinc are the two most important examples—are incorporated into enzymes that catalyze biochemical transformations occurring at key points in the carbon cycle.

The enzyme carbonic anhydrase (CA) catalyzes the interconversion between carbon dioxide—the form of inorganic carbon used by plants in photosynthesis—and bicarbonate, the predominant form of inorganic carbon in sea water. Carbonic anhydrase normally includes zinc at the active site. We have evidence that at the extremely low zinc concentrations of open-ocean surface waters, not enough functional carbonic anhydrase can be made by microalgae; this, in turn, may limit carbon acquisition and, consequently, the rate of algal growth. This limitation is particularly important for diatoms, which are responsible for fixing the bulk of carbon that is exported from surface waters to the deep ocean.

We have found, however, that some species have the capacity to replace zinc with cobalt, and that another form of carbonic anhydrase exists that utilizes cadmium. We recently isolated, sequenced, and expressed a diatom carbonic anhydrase, and obtained in vivo evidence that cobalt replaces zinc in isolated carbonic anhydrase. The cadmium-containing carbonic anhydrase is a different enzyme, and the only known cadmium-containing enzyme in all of nature. We have the opportunity to examine how the carbonic anhydrase of these organisms, whose amino-acid sequence resembles no other known carbonic anhydrase enzyme, functions with a different metal, and how metal substitution occurs.

Our specific objectives are: to further characterize the diatom zinc carbonic anhydrase and study in vitro the Zn/Co replacement; to purify and characterize the putative cadmium carbonic anhydrase; to use genetic and immunological probes to study, in the laboratory and in the field, the regulation of carbonic anhydrase and the possible co-limitation on growth by carbon and zinc.

Zinc/cobalt carbonic anhydrase. We measure Reaction rates and products of both the zinc and cobalt forms of the enzyme using standard catalytic assays. These measurements tell us much about reaction mechanisms and how they vary between the zinc and cobalt versions of the enzyme. We also measure equilibrium constants for metal-ion binding by both Zn²⁺ and Co²⁺ and compare the affinities of the enzyme for binding the two metals.

Cadmium carbonic anhydrase. The cadmium protein is isolated by standard chromatographic methods. Standard genetic methods generate sufficient quantities of the protein for further analysis, including standard catalytic assays. This protein also facilitates development of anti-sera for the cadmium carbonic anhydrase enzyme, to be used for the study of regulation (see below). The enzyme's structure is also studied: the active site is characterized by ¹¹³Cd NMR , and, where appropriate, detailed structural characterization is attempted by X-ray or NMR methods.

Regulation and field studies. We use our existing anti-carbonic-anydrase sera (which react with zinc carbonic anhydrase in all the diatoms we have tested), and the new anti-sera developed for the cadmium-containing carbonic anhydrase, to study CA regulation in diatoms as a function of metal and inorganic carbon availability. These anti-sera allow us to directly measure the amount of CA protein in a sample. We plan to use these sera for a field study in which we will follow the expression of carbonic anhydrase over the course of a diatom bloom in coastal waters. If successful, this work will be extended to open ocean waters, and to other carbonic anhydrase enzymes, once the necessary antibodies have been obtained.

carbon cycle: hydrocarbon metabolism in the environment: alkane oxygenation

Recent reports indicate that alkanes and aromatic hydrocarbons are biodegraded completely and rapidly in both aerobic and anaerobic sediments. In the case of aerobic alkane degradation, we know that the initial step involves the conversion of the alkane to an aliphatic alcohol, in a reaction carried out by a monooxygenase enzyme, but we don't know if this is typical. We don't know how phylogenetically diverse alkane-degrading microorganisms are in the marine environment, or the range of relevant enzymes and mechanisms.

Our objectives are: 1) to identify the principal alkane degrading organisms in sea water; 2) to characterize the family of genes/proteins responsible for degradation processes; 3) to develop probes to identify the species and activities in environmental samples; 4) to purify the enzymes and investigate the mechanisms of hydrocarbon oxidation via catalytic studies.

The most studied alkane-degradation system is that of the terrestrial isolate Pseudomonas oleovorans. Pseudomonas uses a membrane-associated alkane hydroxylase enzyme (AlkB, EC 1.14.15.3) to catalyze the initial degradation of alkanes to the corresponding alcohol. Recent studies indicate that the active site of this alkane hydroxylase enzyme has a dinuclear iron cluster of the type found in soluble diiron proteins such as hemerythrin, ribonucleotide reductase, methane monooxygenase, and other enzymes. The genes encoding this enzyme, and those involved in subsequent steps, have been cloned and expressed in Escherichia coli, yielding a familiar, easy-to-grow bacterium possessing Pseudomonas' hydroxylase enzyme.

To investigate the diversity of alkane-degrading organisms and the role of iron in the oxidation of alkanes in the marine environment, Cebic scientists isolate alkane degraders from sea water under a variety of conditions. These organisms are sorted taxonomically and phenotypically to determine the diversity of organisms involved. The AlkB gene is used to screen isolated organisms for alkane oxidation systems similar to AlkB; the presence of alkane-degradation activity in samples where no genetic match was found indicates a new alkane-degradation mechanism. Based on initial screening studies, organisms covering a range of taxonomic and alkane-oxidation gene types are selected, and the alkane-degradation genes isolated. These genes are sequenced and compared to identify key similarities and differences that may illuminate the structure and functional attributes of the corresponding enzymes. The isolated genes are then used as probes to re-screen the culture collection for additional hydroxylase genes. The set of alkane-oxidation genes obtained in this way are then used to determine their presence and abundance in marine environments.

Cebic scientists are studying the enzymatic mechanisms of alkane oxidation for the monooxygenases obtained from isolated organisms. The taxonomic identification of the isolates, substrate range, and sequence of the monooxygenases are being used to identify those enzymes that appear most interesting and amenable to study. Those enzymes are purified and the structure of the active sites are characterized, as appropriate. The substrate specificity of the various proteins is probed, and isotopically labeled substrates are used to obtain mechanistic information from kinetic isotope effects. AlkB is the first hydroxylase to be studied, to establish a common basis for comparison for newly discovered proteins.

Cebic scientists have also begun to study recently discovered anaerobic alkane degraders. We have two model strains (a Desulfobacterium oleovorans from culture collection and a Desulfosarcina-like strain (AK01) isolated from sediment) that appear to possess distinct mechanisms of anoxic alkane degradation. This contrast provides us with a unique opportunity to determine the degradation pathway and the responsible genes, to establish structural and functional relationships between alkane oxidation enzyme groups, and to determine the role each group plays in the marine environment.

Nitrogen cycle: denitrification (dissimilatory nitrate reduction)

Denitrification is an important chemical transformation in the nitrogen cycle and the principal sink for fixed nitrogen in natural waters. Denitrification occurs as a result of the dissimilatory reduction of nitrogen oxides (nitrate and nitrite), in which nitrogen oxides are used as alternative electron acceptors during respiration by anaerobic bacteria. Denitrification involves a suite of reductase enzymes, all of which require metal cofactors. We shall focus on the nitrate and nitrite reductases.

Dissimilatory nitrate reductases, which contain molybdenum and iron, are usually membrane-bound and oxygen-sensitive. Two major forms of dissimilatory nitrite reductase are found in denitrifying bacteria, one containing iron (heme cd1) and one containing copper (type I and type II); both forms have been crystallographically characterized. Bacterial isolates suggest that the iron-containing enzyme is more common, but methods to assess the abundance in nature of these enzymes and their genes are just being developed.

The major objectives of this project are to establish the relationships between the availability of metals (molybdenum, iron, copper), the diversity and efficiency of enzymes (nitrate and nitrite reductases), and the structure and mechanisms of the enzymes' metal centers. In particular, we wish to understand how the relative availabilities of iron and copper influence the distribution of the different types of nitrite reductase in the environment, and the efficiency of denitrification.

Cebic begins by investigating the diversity, distribution, and abundance in natural system of the genes encoding nitrogen-reducing enzymes. This can be done using techniques similar to those described in the section on siderophore uptake: we search for genes in natural samples that resemble known reductase genes. This requires isolating genes for the nitrogen-reducing enzymes in cultured strains ("probes"), obtaining homologous fragments of DNA from environmental samples, sequencing those fragments, making more primers and probes, and developing quantitative hybridization (or PCR) assays for detecting those genes in the environment. Using the resulting DNA sequences, we will express the protein in a suitable vector and perform structural and functional characterizations on the resulting organism.

Once these genetic methods have been developed, we can study how the abundance in field samples of different nitrate reductase genes (particularly the iron and copper forms) varies with changes in nitrogen and trace-metal availability.

Nitrate acquisition (assimilatory nitrate reduction)

The use of nitrate in biosynthesis requires chemical reduction to the level of ammonium. This reduction is catalyzed by assimilatory nitrate and nitrite reductase enzymes. Recent field work has shown that nitrate reductase activity is limited by iron availability. Our objective is to elucidate the relation between trace-element physiology and the activity of nitrate and nitrite reductases in marine autotrophs.

This project follows the methodology of the previous project (with the advantages of hindsight). In addition, we are studying the cellular cycling of molybdenum and its correlation with the synthesis and degradation of nitrate reductase. Specifically, we wish to know what happens to molybdenum during nitrate-reductase degradation in the dark, and how it is retained within the cell. We hope to gain insight into this process in preliminary experiments using a recently constructed nitrate-reductase-replete strain of Chlamydomonas (the cultured strains of which are usually nitrate-reductase-deficient). This strain has been engineered to resist the antibacterial spectromycin. This antibiotic resistance will make it easy to obtain pure cultures.

Nitrogen cycle: nitrogen fixation

In marine systems, nitrogen is fixed primarily by a single genus of cyanobacteria, Trichodesmium. These organisms are unusual in that they fix N₂ and produce O₂ from photosynthesis within the same cell at the same time. The nitrogenase in Trichodesmium is presumed to contain molybdenum and a lot of iron, as it does in other cyanophytes. Because of the massive iron requirement of the nitrogenase enzyme, some scientists have hypothesized that N₂-fixation in oceans may be limited globally by iron. Two Trichodesmium strains are presently in culture in Cebic laboratories, and the genes for several of the subunits of the multimeric nitrogenase complex have been sequenced.

Our objectives are to examine in Trichodesmium: (1) the relation between the availability of metals (Fe, Mo, V) and N₂-fixation; and (2) the mechanism of protection of nitrogenase from inactivation by O₂. We hypothesize that the nitrogenase is protected by the scavenging of O₂ by an unusually high level of photosynthetic activity.

For the first task, we are measuring, in laboratory cultures, the effect on N₂-fixation rates of modifying the trace-metal chemistry in the sea-water medium. For the second, we shall use knock-out mutations of photosystem-I genes. The relative activities of PS(I) and nitrogenase will be compared in the wild type and the mutant.

Next project: Modeling
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