Functional evolution of biosynthetic enzymes that produce plant volatiles*

Plants synthesize volatile compounds to attract pollinators. The volatiles emitted by flowers are often complex mixtures of organic compounds; pollinators are capable of distinctly recognizing different volatile compounds. Plants also produce volatile compounds to protect themselves against herbivores and pathogens. Some of the volatile compounds produced in floral and vegetative tissues are toxic to insects and microbes. To adapt changes in the environment, plants have evolved the ability to synthesize a unique set of volatiles. Intensive studies have identified and characterized the enzymes responsible for the formation of plant volatiles. In particular, many biosynthetic genes have been isolated and their enzymatic functions have been proposed. This review describes how plants have evolved the biosynthetic pathways leading to the formation of green leaf volatiles and phenylpropene volatiles.     

Particulate guanylyl cyclases: multiple mechanisms of activation

Cyclic GMP (cGMP), a key messenger in several signal transduction pathways, is synthesized from GTP by a family of enzymes termed guanylyl cyclases, which are found in two forms: cytosolic (soluble) and membrane-bound (particulate). The past decade has brought significant progress in understanding the molecular mechanisms that underlie the regulation of particulate guanylyl cyclases and new members of their family have been identified. It has become more evident that the basic mechanism of catalysis of guanylyl cyclases is analogous to that recognized in adenylyl cyclases. Here we review the known basic mechanisms that contribute to the regulation of particulate guanylyl cyclases.     

Cyclic ADP-ribose: A calcium mobilizing metabolite of NAD+

Mobilization of Ca⁺² from intracellular stores is a signalling mechanism that is of fundamental importance to many cellular processes. It is mediated by two major mechanisms, the inositol 1,4,5-trisphosphate pathway and the Ca⁺²-induced Ca⁺² release process. A naturally occurring metabolite of NAD⁺ called cyclic ADP-ribose has been discovered recently and shown to be as effective as inositol 1,4,5-trisphosphate in mobilizing Ca⁺² stores in sea urchin eggs, a marine invertebrate cell, as well as several mammalian cells. This article reviews the accumulating evidence that indicates cyclic ADP-ribose may function as a physiological regulator of the Ca⁺²-induced Ca⁺² release process and the current knowledge about its receptor as well as the enzymes involved in its metabolism.     

Structure and activity of enzymes that remove histone modifications

The post-translational modification of histones plays an important role in chromatin regulation, a process that insures the fidelity of gene expression and other DNA transactions. Equally important as the enzymes that generate these modifications are the enzymes that remove them. Recent studies have identified some of the enzymes that remove histone modifications and have characterized their activities. In addition, structural and biochemical studies of these enzymes have focused on the histone lysine deacetylases HDAC8 and sirtuins, and on the arginine and lysine demethylases PAD and BHC110/LSD1, respectively. These new findings may be used as a context to present new information that contributes to our understanding of chromatin regulation, and to pose remaining questions pertaining to the activities of these enzymes and the roles they play in chromatin regulation.     

Structural enzymology of carbohydrate-active enzymes: implications for the post-genomic era

Simple and complex carbohydrates have been described as "the last frontier of molecular and cell biology". The enzymes that are required for the synthesis and degradation of these compounds provide an enormous challenge in the post-genomic era. This reflects both the extreme chemical and functional diversity of sugars and the difficulties in characterizing both the substrates and the enzymes themselves. The vast myriad of enzymes involved in the synthesis, modification and degradation of oligosaccharides and polysaccharides is only just being unveiled by genomic sequencing. These so-called "carbohydrate-active enzymes" lend themselves to classification by sensitive sequence similarity detection methods. The modularity, often extremely complex, of these enzymes must first be dissected and annotated before high throughput characterization or "structural genomics" approaches may be employed. Once achieved, modular analysis also permits collation of a detailed "census" of carbohydrate-active enzymes for a whole organism or throughout an ecosystem. At the structural level, improvements in X-ray crystallography have opened up a three-dimensional understanding of the way these enzymes work. The mechanisms of many of the glycoside hydrolase families are becoming clearer, yet glycosyltransferases are only slowly revealing their secrets. What is clear from the genomic and structural data is that if we are to harness the latent power of glycogenomics, scientists must consider distant sequence relatives revealed by the sequence families or other sensitive detection methods.     

Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.     

Fungi that produce mycotoxins: Conditions and occurrence

The occurrence of mycotoxins, in agricultural commodities is a worldwide problem with almost all commodities being potentially susceptible to contamination under the proper conditions. The genera of fungi most implicated are Aspergillus, Penicillium and Fusarium, although the potential for toxin production varies considerably within any given species.Conditions that affect toxin production include fungal strain variation, genetic susceptibility of the host plant or commodity, moisture content, commodity composition, temperature, aeration, microbial population and stress factors. There is undoubtedly interaction between these factors so that laboratory studies involving limited variables can only, at best, approximate field conditions.Natural contamination with mycotoxins has been reported for most of the major agricultural commodities in the world including corn, wheat, rice, millet, barley, oats, sorghum, peanuts, beans, copra, some fruits and nuts and various forages; strangely, soybeans do not appear to be involved to any major extent. The major mycotoxins on commodities reported to date include aflatoxin, the trichothecenes, ochratoxin, citrinin, zearalenone, sporidesmins and some tremorgens. However, laboratory studies have shown that the fungi are capable of producing hundreds of toxic chemicals, most of which are not included in routine analyses. In addition, since toxin effects are often insidious and may go undetected, the true dimensions of the mycotoxin problem are unknown.     

Catalytic function of the residues of phenylalanine and tyrosine conserved in squalene-hopene cyclases.

Site-directed mutagenesis experiments on all the conserved residues of Phe and Tyr in all the known squalene-hopene cyclases (SHCs) were carried out to identify the active site residues of thermophilic Alicyclobacillus acidocaldarius SHC. The following functions are proposed on the basis of kinetic data and trapping of the prematurely cyclized products: (1) The Y495 residue probably amplifies the D376 acidity, which is assumed to work as a proton donor for initiating the polycyclization cascade, but its role is moderate. (2) Y609 possibly assists the function of F365, which has previously been assigned to exclusively stabilize the C-8 carbocation intermediate through cation-pi interaction. The Y609A mutant produced a partially cyclized bicyclic triterpene. (3) Y612 works to stabilize both the C10 and C8 carbocations, this being verified by the finding that mono- and bicyclic products were formed with the Y612A mutant. (4) F129 was first identified to play a crucial role in catalysis. (5) The three residues, Y372, Y474 and Y540, are responsible for reinforcing the protein structure against thermal denaturation, Y474 being located inside QW motif 3.     

Molecular cloning, expression, and functional characterization of a novel member of the CD38 family of ADP-ribosyl cyclases

We report the molecular cloning and functional characterization of a novel member of the CD38 family of cyclic ADP-ribose (cADPr)-generating cyclases. We cloned a cDNA insert that encoded a 298-amino-acid-long protein (M(w) approximately 39 kDa). The predicted protein displayed 69, 61, and 58% similarity, respectively, to mouse, rat, and human CD38. Rabbit CD38 was also 28% homologous to Aplysia ADP-ribosyl cyclase and leukocyte CD157 (another ADP-ribosyl cyclase); the three cyclases shared 10 cysteine and 2 adjacent proline residues. We then transfected CD38-negative NIH3T3 cells with cDNA encoding a CD38-EGFP fusion protein. Epifluorescence microscopy showed intense EGFP fluorescence confirming CD38 expression. We finally confirmed the ADP-ribosyl cyclase activity of the expressed CD38 by measuring its ability to catalyze the cyclization of the nicotinamide adenine dinucleotide (NAD(+)) surrogate, NGD(+), to its fluorescent nonhydrolyzable derivative, cGDPr.     

Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species

NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hormone synthesis. In conjunction with NADPH oxidation, Nox enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. The electron-transferring system in Nox is composed of the C-terminal cytoplasmic region homologous to the prokaryotic (and organelle) enzyme ferredoxin reductase and the N-terminal six transmembrane segments containing two hemes, a structure similar to that of cytochrome b of the mitochondrial bc(1) complex. During the course of eukaryote evolution, Nox enzymes have developed regulatory mechanisms, depending on their functions, by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit. For example, one to four Ca(2+)-binding EF-hand motifs are present at the N-termini in several subfamilies, such as the respiratory burst oxidase homolog (Rboh) subfamily in land plants (the supergroup Plantae), the NoxC subfamily in social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox) subfamilies in animals (the Opisthokonta), whereas an SH3 domain is inserted into the ferredoxin-NADP(+) reductase region of two Nox enzymes in Naegleria gruberi, a unicellular organism that belongs to the supergroup Excavata. Members of the Nox1-4 subfamily in animals form a stable heterodimer with the membrane protein p22(phox), which functions as a docking site for the SH3 domain-containing regulatory proteins p47(phox), p67(phox), and p40(phox); the small GTPase Rac binds to p67(phox) (or its homologous protein), which serves as a switch for Nox activation. Similarly, Rac activates the fungal NoxA via binding to the p67(phox)-like protein Nox regulator (NoxR). In plants, on the other hand, this GTPase directly interacts with the N-terminus of Rboh, leading to superoxide production. Here I describe the regulation of Nox-family oxidases on the basis of three-dimensional structures and evolutionary conservation.     

Class III nucleotide cyclases in bacteria and archaebacteria: lineage-specific expansion of adenylyl cyclases and a dearth of guanylyl cyclases

The Class III nucleotide cyclases are found in bacteria, eukaryotes and archaebacteria. Our survey of the bacterial and archaebacterial genome and plasmid sequences identified 193 Class III cyclase genes in only 29 species, of which we predict the majority to be adenylyl cyclases. Interestingly, several putative cyclase genes were found to have non-conserved substrate specifying residues. Ancestors of the eukaryotic C1-C2 domain containing soluble adenylyl cyclases as well as the protist guanylyl cyclases were found in bacteria. Diverse domains were fused to the cyclase domain and phylogenetic analysis indicated that most proteins within a single cluster have similar domain compositions, emphasising the ancient evolutionary origin and versatility of the cyclase domain.     

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4⁺ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.     

Escherichia coli has multiple enzymes that attack TNT and release nitrogen for growth

There has been a growing interest in the degradation of 2,4,6-trinitrotoluene (TNT) over the last decade, ever since its removal from polluted sites was declared an international environmental priority. Certain aerobic and anaerobic microorganisms are capable of using TNT as an N source, although very few studies have proven the mineralization of this compound. An unexpected observation in our laboratory led us to discover that certain Escherichia coli bench laboratory strains have multiple enzymes that attack TNT. One of the NemA products is responsible for the release of nitrite from the nitroaromatic ring: among the metabolites observed in vitro include Meisenheimer dihydride complexes of TNT from which 2-hydroxylamino-6-nitrotoluene is slowly formed during their rearomatization under concomitant release of nitrite. Furthermore, NemA, together with NfsA and NfsB reduce the nitro groups on the aromatic ring to the corresponding hydroxylamino derivatives, which probably results in the release of ammonium ions which can, in turn be used as a nitrogen source by E. coli for growth.     

Structure, function and evolution of microbial adenylyl and guanylyl cyclases

Cells respond to signals of both environmental and biological origin. Responses are often receptor mediated and result in the synthesis of so-called second messengers that then provide a link between extracellular signals and downstream events, including changes in gene expression. Cyclic nucleotides (cAMP and cGMP) are among the most widely studied of this class of molecule. Research on their function and mode of action has been a paradigm for signal transduction systems and has shaped our understanding of this important area of biology. Cyclic nucleotides have diverse regulatory roles in both unicellular and multicellular organisms, highlighting the utility and success of this system of molecular communication. This review will examine the structural diversity of microbial adenylyl and guanylyl cyclases, the enzymes that synthesize cAMP and cGMP respectively. We will address the relationship of structure to biological function and speculate on the complex origin of these crucial regulatory molecules. A review is timely because the explosion of data from the various genome projects is providing new and exciting insights into protein function and evolution.