Regulation of Steroid BiosynthesisThe plant steroid hormones, brassinosteroids BRsand their precursors, phytosterols, play major roles in plant growth, development, and stress tolerance. Here, we review the impressive progress made during recent years in elucidating the components of the sterol and BR metabolic and signaling pathways, and in understanding their biosynthesis of steroids from squalene of action in both model plants and crops, such as Arabidopsis and rice. We also discuss emerging insights into the regulations of these pathways, their interactions with other hormonal pathways and multiple genotropin hgh for sale signals, and the biosynthesis of steroids from squalene nature of sterols as signaling molecules. The plant steroid hormones, brassinosteroids, and their precursors, phytosterols, are essential for plant growth, reproduction, and response to various abiotic and biotic stresses. We review their biological activities and discuss big gut bulking advances in elucidating their metabolism, transport, and signaling pathways.
Regulation of Steroid Biosynthesis | Annual Review of Biochemistry
Common tetracyclic steroid frame containing the 1,2-cyclopentanoperhydrophenanthrene ring skeleton. The first known sterol, cholesterol, was discovered by French chemists as a crystalline component of human gallstones over years ago. In , Francois Poulletier de La Salle observed an alcohol-soluble portion of bile stones, which 10 years later was reported by De Fourcroy to be identical to a waxy material in the fat of putrefied corpses referred to as adipocire. The correct formula C 27 H 45 O—, which here shows that it has a hydroxyl group although it contains 46 hydrogen atoms of cholesterol was proposed in by F.
Reinitze, yet it took another 30 years to establish the exact steric representation of the molecule, efforts that led to the Nobel Prizes in chemistry for Wieland and Windaus The first connection between cholesterol and human health appeared in as Vogel showed that cholesterol was present in arterial plaques. Sterol research changed in the mid- to late 20th century and centered on biomimetic chemistry, tracer work, enzymology, and structure determination using high-field NMR and X-ray diffraction methods, culminating with a broad outline for cholesterol synthesis and the partial or complete purification of all the microsomal-bound enzymes that act on sterols between lanosterol and cholesterol.
Central to the advances of the past two decades is the development of molecular genetic approaches that have witnessed the cloning, primary amino acid sequences, and functional characterization of a large number of enzymes that act on sterol and revealed unexpected inborn errors of cholesterol metabolism. The full exploitation of these genes lies in medical diagnostics, treatment, and the ability to ultimately engineer phytosterol pathways to generate plants with tailored sterol profiles for commercial production.
Another spectacular discovery involving 13 C-isotopically labeled compounds supplied to microorganisms and plants was the demonstration that the classic acetate—mevalonate pathway to animal cholesterol can be replaced in the biosynthesis of algal sterols and other isoprenoids by a mevalonate-independent pathway.
Their efforts constitute a collective undertaking of significant importance to several remarkable advances toward the completion of the enzymatic inventory of sterol synthesis, and these new findings, together with a brief examination of prior art in the field of sterols, are discussed in this review.
Accordingly, the sterol molecule possesses four indispensible domains. In domain A, the polarity and tilt of the C3 OH-group contribute functionally to hydrogen-bond interactions.
In domain B, the C4 and methyl groups can affect the A ring conformation and back face planarity, respectively. Alternatively, the number and position of double bonds in the nucleus can affect the shape of the sterol and tilt of the 17 20 -bond. In domain D, the conformation and length of the side chain, in addition to the stereochemistry of the Calkyl group in phytosterols, are critical to intermolecular contacts.
Perspective drawings of the cholesterol molecule showing four domains of functional importance left and the flat elongated structure presumed to form in the membrane right. The earliest chemical definition for a sterol was provided by Fieser and Fieser. The revised system has further complications and becomes cumbersome as the complexity of side-chain modifications by biosynthetic alkylations increase.
Two systems recognized for numbering of carbon atoms of the sterol nucleus and side chain. Because sterols are derived from the C 30 squalene, they are a class of triterpenoids. These tetracycles are generated from the linear combinations of the C 5 -isoprenoid building block, isopentyl diphosphate.
For this alternate definition, the focus is on the reaction mechanism and ignores the precise isoprenoid character of the cyclization product, assuming only that the intermediate adopts steroidal character during cyclization to produce a tetracycle compound of specific structure and stereochemistry.
Thus, a true sterol is formed by the electrophilic cyclization reactions that pass through a transition state similar to the trans — syn — trans — anti — trans — anti configuration affording a protosteroid C20 cation. The cyclization of squalene-2,3-oxide i folded in either the chair—chair—chair—boat—unfolded ii or chair—boat—chair—boat—unfolded ii conformations to yield a cation ii , which can stabilize to produce the dammarane, tirucallane, euphane, and cucurbitane skeletons via path a or cycloartane and lanostane skeletons via path b.
Chemical surveys of the sterol composition of prokaryotes, eukaryotes, and sedimentary organic matter show that there are at least sterols and related steranes; in corn, 60 different sterols have been characterized. Djerassi and his associates, using a computer-assisted program, calculate that natural sterols may have as many as different structures; many of them may be found in marine organisms, which are known to synthesize highly bioalkylated sterol side chains.
According to the currently accepted hypothesis, the formation of steroids proceeds by a cationic cyclization process. This theory has it roots in the biogenetic isoprene rule of Ruzicka and his associates in Zurich who considered biosynthesis of triterpenoids was initiated by an electrophilic attack on a double bond of a linear polyprenoid substrate forming a cyclic or polycyclic intermediate cation, which, in turn, can then undergo various transformations and rearrangements.
Ionization of the allylic pyrophosphate leads to formation of a charge-stabilized allylic cation. Two structurally unrelated classes of isopentyl diphosphate isomerase IDI are known.
In contrast, the type II enzyme IDI-2 requires reduced flavin, raising the possibility that the reaction catalyzed by IDI-2 involves the net addition or abstraction of a hydrogen atom. The prenyl transferase reaction and formation of farnesyl diphosphate FPP. Alternate isoprene unit assemblies yield a variety of different structures notable in terpene metabolism.
The biosynthesis of C 30 sterols from squalene and thence to cholesterol can be outlined in three major stages as envisioned by Bloch: Two molecules of farnesyl diphosphate condense tail to tail to the C 30 acyclic polyene squalene by the action of squalene synthase SQS. The C 30 symmetric olefin undergoes oxidation to form S -oxidosqualene via an NADPH-dependent mono-oxygenase reaction catalyzed by squalene epoxidase SQE , and this substrate can be cyclized by an oxidosqualene—sterol synthase to yield the steroidal backbone structure represented in lanosterol.
In stage 3, lanosterol is converted to cholesterol section. In the early phase of cholesterol research, it was not immediately apparent that the C 27 structure of cholesterol was related to lanosterol, since it failed to be divisible by C 5 units.
To establish isoprenoid character, several groups incubated [1- 14 C]acetate and [2- 14 C]acetate with liver slices affording a decisive pattern in the distribution of acetate carbon atoms in the labeled cholesterol. These tracer studies also provided the foundation for the acetate—mevalonate pathway in sterol biosynthesis. Distribution of acetate carbon atoms found in cholesterol; a repeating pattern of five carbon atoms isoprene unit , surrounded by dotted lines, is recognizable in three places in the molecule.
Since the late s, it has been known that two major cyclization pathways exist for the conversion of oxidosqualene to steroidal tetracycles; lanosterol is formed in organisms of a nonphotsynthetic lineage, and cycloartenol is formed in organisms of a photosynthetic lineage by independent synthase enzymes. Incubation of oxidosqualene bearing a chiral methyl group H, 2 H, 3 H at C6 with the plant synthase revealed that the stereochemistry of the cyclopropane ring closure proceeds with retention of configuration.
If this is not done, the final step, migration of a hydrogen from Cmethyl, will be cis to the C9 hydrogen transfer instead of trans in order to conform with the biogenetic isoprene rule. Subsequent withdrawal of the X — then permits closure of the cyclopropane ring in a trans manner with concomitant removal of the C19 proton, which is tantamount to a double inversion mechanism.
These postulates are revisited in section. Interpretation of the mechanism of squalene-2,3-oxide cyclization to lanosterol and cycloartenol according to refs 61 , 63 , and The different molecular libraries that constitute isoprenoid—sterol metabolomes across Kingdoms are organized through a series of discrete assemblies of enzymatic reactions, which are characterized compartmentally. The acetate—MVA pathway to squalene oxide is considered to be the main route to the production of steroidal backbones.
Recent international efforts have resulted in the complete sequencing of the model plant Arabidopsis , nonpathogenic fungus Saccharomyces , and human genomes. The functional genomics approach together with the establishment of defective biosynthesis steps in humans and generation of yeast and plant mutants in sterol biosynthesis enabled the elucidation of structural genes for the individual enzymes in sitosterol, ergosterol, and cholesterol formation.
In nonanimal systems different sterolic genes can encode for similar reaction steps, for example, sterol Cmethyltransferase, sterol Cdemethylase, or sterol methyl oxidase, whereas mammals generally have only a single gene for each enzymatic step. The best described gene—gene product pairing is in the yeast pathway for which every relevant gene in the conversion of lanosterol to ergosterol has been identified. The principal enzymes of lanosterol conversion to cholesterol are coded for by nine genes section.
Isoforms can exist to alter the number of enzymatic reactions in a specific pathway. The other, known as the methyl- d -erythritol 4-phosphate MEP pathway or mevalonate-independent pathway, utilizes triose phosphate units as the precursor and is located in the plastid of plants.
Many of these enzymes have been characterized structurally. Terpenoids of the C 10 , C 15 , C 20 , and C 40 skeletons are generally synthesized within the plastid and sterols synthesized in the cytosol of higher plants. Goad and co-workers reported that mitochondria of some parasitic protozoa have the capability to convert leucine to MVA, which then converts to IPP and ergosterol via the cytosol.
Overview of compartmentalized isoprenoid—sterol biosynthesis pathways. The 13 C-labeling pattern of ergosterol synthesized in all major eukaryote Kingdoms reveals which of the pathways operate in a given organism and whether cross-talk between the pathways exist.
It has been possible to establish the contribution of these variant isoprenoid pathways to sterol biosynthesis in plants and microorganisms by the retrobiosynthetic approach. In the diatom Rhizosolenia setigera that can synthesize ethyl sterol, the MEP pathway is favored.
By far the best studied of the enzymes involved in sterol biosynthesis are the lanosterol and cycloartenol synthases. For cyclization, the reaction requires 3 S -2,3-oxidosqualene first to adopt a preorganized chair—boat—chair conformation. The cyclization of squalene-2,3-oxide 1 catalyzed by the lanosterol synthase LAS or cycloartenol synthase CAS to give true sterols.
Examination of yeast ERG 7 homology structure and human OSC structure suggest that Tyr and Tyr 99 residues in the Saccharomyces cerevisiae ScERG 7 might play an important role in stabilizing the C8 cation during the formation of the second cyclohexyl ring and the final lanosterol C9 cation.
Although cycloartenol is the first cyclized product in plants, lanosterol has been considered as an intermediate in phytosterol biosynthesis. However, primary sequences of lanosterol synthase were discovered recently in three different laboratories from dicotyledonous plant species, including Arabidopsis thaliana , Panax ginseng , and Lotus japonica , using a yeast expression system that suggest that lanosterol can be synthesized directly and independently from cycloartenol.
Three different approaches have been used to study the nature and sequence of steps involved with sterol biosynthesis: The enzymatic approach to understand and control formation of the sterol structure was hampered by the low abundance of sterol enzymes in cell-free preparations, as well as difficulties associated with purifying microsomal proteins to homogeneity. This process can be divided into two stages: During these conversions of the C4-sterol to a C4-desmethyl sterol, a stable 3-keto sterol intermediate is formed.
Key to this series of reactions is that the equatorial in the plane of the sterol nucleus methyl group of the 4,4-dimethyl and 4-monomethyl substrates is recognized for catalysis.
Two enzymatic studies completed on the cloned human sterol C8—C7 isomerase and microsomal rat C24 25 -reductase after Gaylor postulated a lanosterol—cholesterol pathway clarified the later sequence of reactions to cholesterol.
As more enzymes have been isolated from an increasing number of sources, it has become clear that they fall into only a small number of reaction types and that the chemical names give varied indication of this. The most direct route to cholesterol will be dependent upon the relative specificities of the enzymes for a particular sterol substrate thereby giving rise to the kinetically favored pathway.
When multiple routes are postulated, it has been possible to draw a sterol biosynthesis matrix for an organism, which can predict the main or trace sterols which might be present, particularly after exposure to a sterol biosynthesis inhibitor or genetic defect. In the case of cholesterol biosynthesis from lanosterol, two intersecting routes have been postulated.
The choice of pathway is determined by the stage at which the double bond at C24 in the sterol side chain is reduced. If C24 double bond reduction is retained until the last reaction, cholesterol synthesis proceeds via cholesta-5,dienol desmosterol Bloch pathway. The relevant committed step that distinguishes sterol from isoprenoid—triterpenoid biosynthesis occurs at the cyclization of oxidosqualene. Major control points in sterol biosynthesis may arise in the primary pathway before squalene formation at hydroxymethyl-glutaryl-CoA reductase HMGR coarse control 3 or after squalene formation at the sterol Cmethyltransferase SMT step fine control specific to organisms other than animals.
Cofactor control by differential allocation of oxygen, NADPH, and AdoMet can further influence reaction rates and product distributions in cholesterol or phytosterol biosynthesis. Early attempts to deduce sterol biosynthetic pathways in systems other than animals were based largely on indirect approaches that included structural and stereochemical correlations of co-occurring metabolites and in vivo tracer studies. Using microsome preparations of corn, Rahier, Benveniste, and their co-workers systematically worked through the major enzymatic reactions from cycloartenol to sitosterol and established substrate preference for many of these enzymes.
The sterol alkylation reaction pathways operating in different organisms; routes 1 in fungi, protozoa, and plants to form C 9 and C 10 side chains and routes 2 and 3 to form C 11 extended side chains in marine organisms. In organisms that live in the marine world, sterol methylation patterns vary greatly and reflect the complexity of mixtures of sterols arising through the food chain. Whereas plants can synthesize as many as 60 sterols, marine organisms have been shown to contain as many as 74 sterols in a single organism.
The stereochemistry of the transmethylation reactions and subsequent side chain modifications by reduction have been subjected to detailed investigation. In reviewing the action of enzymes that catalyze sterol formation, it has been found convenient to divide topics according to the properties of the enzyme that include its specificity, mechanism, inhibition, and where evidence is available results of mutagenesis experiments. We start with the enzymatic Cmethylation reaction, where progress has been made in identifying the genes and catalytic properties of the corresponding proteins.
Four different sterol Cmethyltransferase enzymes SMT have been detected and classified according to the substrate favored by the enzyme for catalysis.
An interesting feature of several of the cloned SMTs is their ability to produce multiple and distinct product sets at either the C 1 - or C 2 -stage of methylation. Twenty five of these residues lie in the substrate binding segments, and another 38 lie outside of these areas.
Steady-state kinetic studies show fungal SMT1 S. Alternatively, the plant SMT1 G. The structure of SMT has not been established.