Molecular enzymology of 5-Aminolevulinate synthase, the gatekeeper of heme biosynthesis

Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612-4799, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 11/2011; 1814(11):1467-73. DOI: 10.1016/j.bbapap.2010.12.015
Source: PubMed


Pyridoxal-5'-phosphate (PLP) is an obligatory cofactor for the homodimeric mitochondrial enzyme 5-aminolevulinate synthase (ALAS), which controls metabolic flux into the porphyrin biosynthetic pathway in animals, fungi, and the α-subclass of proteobacteria. Recent work has provided an explanation for how this enzyme can utilize PLP to catalyze the mechanistically unusual cleavage of not one but two substrate amino acid α-carbon bonds, without violating the theory of stereoelectronic control of PLP reaction-type specificity. Ironically, the complex chemistry is kinetically insignificant, and it is the movement of an active site loop that defines k(cat) and ultimately, the rate of porphyrin biosynthesis. The kinetic behavior of the enzyme is consistent with an equilibrium ordered induced-fit mechanism wherein glycine must bind first and a portion of the intrinsic binding energy with succinyl-Coenzyme A is then utilized to perturb the enzyme conformational equilibrium towards a closed state wherein catalysis occurs. Return to the open conformation, coincident with ALA dissociation, is the slowest step of the reaction cycle. A diverse variety of loop mutations have been associated with hyperactivity, suggesting the enzyme has evolved to be purposefully slow, perhaps as a means to allow for rapid up-regulation of activity in response to an as yet undiscovered allosteric type effector. Recently it was discovered that human erythroid ALAS mutations can be associated with two very different diseases. Mutations that down-regulate activity can lead to X-linked sideroblastic anemia, which is characterized by abnormally high iron levels in mitochondria, while mutations that up-regulate activity are associated with X-linked dominant protoporphyria, which in contrast is phenotypically identified by abnormally high porphyrin levels. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

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    • "The complex outer membrane of Gram-negative bacteria acts as a barrier that hinders the PS to transport through the cell membranes; hence, Gram-negative bacteria appear to be less sensitive to the lethal action of PDI with exogenously supplied porphyrins [7e9]. 5-Aminolevulinic acid (ALA) is a naturally occurring intermediate in the hemesynthesis pathway [10]. It is a precursor of porphyrins that can be biosynthesized in nearly all aerobic cells in mammals. "
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    ABSTRACT: The aim of the present study was to develop a simple and fast screening technique to directly evaluate the bactericidal effects of 5-aminolevulinic acid (ALA)-mediated photodynamic inactivation (PDI) and to determine the optimal antibacterial conditions of ALA concentrations and the total dosage of light in vitro. The effects of PDI on Staphylococcus aureus and Pseudomonas aeruginosa in the presence of various concentrations of ALA (1.0 mM, 2.5 mM, 5.0 mM, 10.0 mM) were examined. All bacterial strains were exponentially grown in the culture medium at room temperature in the dark for 60 minutes and subsequently irradiated with 630 ± 5 nm using a light-emitting diode (LED) red light device for accumulating the light doses up to 216 J/cm2. Both bacterial species were susceptible to the ALA-induced PDI. Photosensitization using 1.0 mM ALA with 162 J/cm2 light dose was able to completely reduce the viable counts of S. aureus. A significant decrease in the bacterial viabilities was observed for P. aeruginosa, where 5.0 mM ALA was photosensitized by accumulating the light dose of 162 J/cm2. We demonstrated that the use of microplate-based assays—by measuring the apparent optical density of bacterial colonies at 595 nm—was able to provide a simple and reliable approach for quickly choosing the parameters of ALA-mediated PDI in the cell suspensions.
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    • "The first committed step of heme biosynthesis in non-plant eukaryotes and some prokaryotes, the pyridoxal 5′-phosphate (PLP)-dependent condensation of glycine and succinyl-coenzyme A to generate 5-aminolevulinate (ALA), coenzyme A (CoA), and CO2, is catalyzed by 5-aminolevulinate synthase (ALAS) [1], [2]. This reaction is directly coupled to the citric acid cycle via the substrate succinyl-CoA and is the key regulatory step of heme biosynthesis [3]. "
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    ABSTRACT: 5-Aminolevulinate synthase (ALAS; EC catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2) is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX), the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21-22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. The delivery of stable and highly active ALAS2 variants has the potential to expand and improve upon current PDT regimes.
    PLoS ONE 04/2014; 9(4):e93078. DOI:10.1371/journal.pone.0093078 · 3.23 Impact Factor
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    • "At present only the crystal structure of ALAS from the bacterium Rhodobacter capsulatus has been determined [20], but it is clear that the mature eukaryotic ALAS is highly similar to the bacterial enzyme except that it possesses an additional carboxyl-terminal sequence of approximately 25 residues . Much is known about the mammalian enzyme from kinetic and site-directed mutagenesis studies [17]. In vertebrates, two isozymes of Biochimica et Biophysica Acta 1823 (2012) 1617–1632 ☆ This article is part of a Special Issue entitled: Cell Biology of Metals. "
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    ABSTRACT: The appearance of heme, an organic ring surrounding an iron atom, in evolution forever changed the efficiency with which organisms were able to generate energy, utilize gasses and catalyze numerous reactions. Because of this, heme has become a near ubiquitous compound among living organisms. In this review we have attempted to assess the current state of heme synthesis and trafficking with a goal of identifying crucial missing information, and propose hypotheses related to trafficking that may generate discussion and research. The possibilities of spatially organized supramolecular enzyme complexes and organelle structures that facilitate efficient heme synthesis and subsequent trafficking are discussed and evaluated. Recently identified players in heme transport and trafficking are reviewed and placed in an organismal context. Additionally, older, well established data are reexamined in light of more recent studies on cellular organization and data available from newer model organisms. This article is part of a Special Issue entitled: Cell Biology of Metals.
    Biochimica et Biophysica Acta 05/2012; 1823(9):1617-32. DOI:10.1016/j.bbamcr.2012.04.009 · 4.66 Impact Factor
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