ALS resistance
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The acetolactate synthase (ALS) enzyme (also known as acetohydroxy acid or acetohydroxyacid synthase, abbr. AHAS) is a protein found in plants and micro-organisms. ALS catalyzes the first step in the synthesis of the
branched-chain amino acids A branched-chain amino acid (BCAA) is an amino acid having an aliphatic side-chain with a branch (a central carbon atom bound to three or more carbon atoms). Among the proteinogenic amino acids, there are three BCAAs: leucine, isoleucine, and va ...
(
valine Valine (symbol Val or V) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- carboxylic acid group (which is in the deprotona ...
,
leucine Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- ...
, and
isoleucine Isoleucine (symbol Ile or I) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depr ...
). A human protein of yet unknown function, sharing some sequence similarity with bacterial ALS, is encoded by the ILVBL (ilvB-like)
gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
.


Structure


Gene

Human ILVBL gene has 17
exons An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding seque ...
resides on
chromosome 19 Chromosome 19 is one of the 23 pairs of chromosomes in humans. People normally have two copies of this chromosome. Chromosome 19 spans more than 58.6 million base pairs, the building material of DNA. It is considered the most gene-rich chromos ...
at q13.1.


Protein

The catalytic peptide of ALS in ''
Arabidopsis thaliana ''Arabidopsis thaliana'', the thale cress, mouse-ear cress or arabidopsis, is a small flowering plant native to Eurasia and Africa. ''A. thaliana'' is considered a weed; it is found along the shoulders of roads and in disturbed land. A winter ...
'' (mouse-eared cress) is a
chloroplast A chloroplast () is a type of membrane-bound organelle known as a plastid that conducts photosynthesis mostly in plant and algal cells. The photosynthetic pigment chlorophyll captures the energy from sunlight, converts it, and stores it i ...
ic
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
consisting of 670 residues, the last 615 of which form the active form. Three main domains are found, with two
thiamine pyrophosphate Thiamine pyrophosphate (TPP or ThPP), or thiamine diphosphate (ThDP), or cocarboxylase is a thiamine (vitamin B1) derivative which is produced by the enzyme thiamine diphosphokinase. Thiamine pyrophosphate is a cofactor that is present in all liv ...
sandwiching a DHS-like NAD/FAD-binding domain. In SCOP assignment, these subunits are named d1yhya1, d1yhya2, and d1yhya3 from the N-terminal to the C-termianl. The structure of acetolactate synthase that was used for the picture on this page was determined using X-ray diffraction at 2.70 angstroms. X-ray diffraction uses X-rays at specified wavelengths to produce patterns, as the X–ray is scattered in certain ways that give an idea to the structure of the molecule being analyzed. There are five specific ligands that interact with this protein. The five are listed below. The FAD bound is not catalytic.


Function

Acetolactate synthase is catalytic
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
involved in the biosynthesis of various amino acids. This enzyme has the Enzyme Commission Code is 2.2.1.6, which means that the enzyme is a transketolase or a transaldolase, which is classified under the transferases that transfer aldehyde or ketone residues. In this case, acetolactate synthase is a transketolase, which moves back and forth, having both catabolic and anabolic forms. These act on a ketone (
pyruvate Pyruvic acid (CH3COCOOH) is the simplest of the alpha-keto acids, with a carboxylic acid and a ketone functional group. Pyruvate, the conjugate base, CH3COCOO−, is an intermediate in several metabolic pathways throughout the cell. Pyruvic a ...
) and can go back and forth in the metabolic chain. These are found in humans, animals, plants, and bacteria. In plants, they are located in the chloroplasts in order to help with the metabolic processes. In baker's yeast, they are located in the mitochondria. In several experiments, it has been shown that mutated strains of Escherichia coli K-12 without the enzyme were not able to grow in the presence of only acetate or oleate as the only carbon sources. A catabolic version that does not bind FAD () is found in some bacteria.


Catalytic activity

Acetolactate synthase, also known as acetohydroxy acid synthase, is an enzyme specifically involved in the conversion of pyruvate to acetolactate: : 2 CH3COCO2O2CC(OH)(CH3)COCH3 + CO2 The reaction uses thiamine pyrophosphate in order to link the two pyruvate molecules. The resulting product of this reaction, acetolactate, eventually becomes valine, leucine, and isoleucine. All three of these amino acids are
essential amino acid An essential amino acid, or indispensable amino acid, is an amino acid that cannot be synthesized from scratch by the organism fast enough to supply its demand, and must therefore come from the diet. Of the 21 amino acids common to all life form ...
s and cannot be synthesized by humans. This also leads to the systemic name pyruvate:pyruvate acetaldehydetransferase (decarboxylating). This enzyme is the first of several enzymes in the biosynthesis cycle for leucine and valine, taking the initial pyruvate molecules and starting the conversion from pyruvic acid to the amino acids. The specific residue that is responsible for this is a glycine at position 511 in the protein. This is the one that requires a cofactor of TPP for its function. Four specific residues are responsible for catalytic activity in this enzyme. They are listed here with cofactors required written after. The primary sequence of this protein in
Mouse-ear cress ''Arabidopsis thaliana'', the thale cress, mouse-ear cress or arabidopsis, is a small flowering plant native to Eurasia and Africa. ''A. thaliana'' is considered a weed; it is found along the shoulders of roads and in disturbed land. A winter a ...
is listed below. Residues involved in catalytic activity are bolded. Mutagenesis of Asp428, which is crucial carboxylate ligand to Mg(2+) in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg(2+). While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg(2+), D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K(+). Because of inhibition and several factors it is a slow procedure.


Regulation

In the mouse-eared cress, two chains of catalytic ALS () is complexed with two regulatory small subunits (), VAT1 and At2g31810. Such an arrangement is widespread in both bacterial and eukaryotic ALS. The hetromeric structure was demonstrated in E. coli in 1984 and in eukaryotes (''S. cerevisiae'' and ''Porphyra purpurea'') in 1997. Most of the regulatory proteins have an ACT domain () and some of them have a NiKR-like C-terminal (). In bacteria (''E. coli'')), Acetolactate synthase consists of three pairs of isoforms. Each pair includes a large subunit, which is thought to be responsible for
catalysis Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
, and a small subunit for
feedback inhibition An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a s ...
. Each subunit pair, or ALS I, II, and III respectively, is located on its own
operon In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splic ...
, ilvBN, ilvGM and ilvIH (where ilvN regulated ilvB, and vice versa). Together, these operons code for several enzymes involved in branched-chain amino acid biosynthesis. Regulation is different for each operon. The ''ilvGMEDA'' operon encodes the ilvGM (ALS II) pair as well as a
branched-chain-amino-acid transaminase Branched-chain amino acid aminotransferase (BCAT), also known as branched-chain amino acid transaminase, is an aminotransferase enzyme which acts upon branched-chain amino acids (BCAAs). It is encoded by the ''BCAT2'' gene in humans. The BCAT enz ...
(ilvE),
dihydroxy-acid dehydratase The enzyme dihydroxy-acid dehydratase () catalyzes the chemical reaction :2,3-dihydroxy-3-methylbutanoate \rightleftharpoons 3-methyl-2-oxobutanoate + H2O This enzyme participates in valine, leucine and isoleucine biosynthesis and pantothenate a ...
(ilvD), and
threonine ammonia-lyase Threonine ammonia-lyase (EC 4.3.1.19, systematic name L-threonine ammonia-lyase (2-oxobutanoate-forming), also commonly referred to as threonine deaminase or threonine dehydratase, is an enzyme responsible for catalyzing the conversion of L-threo ...
(ilvA). It is regulated by
feedback inhibition An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a s ...
in the form of
transcriptional attenuation In genetics, attenuation is a regulatory mechanism for some bacterial operons that results in premature termination of transcription. The canonical example of attenuation used in many introductory genetics textbooks, is ribosome-mediated attenuat ...
. That is, transcription is reduced in the presence of the pathway's end-products, the branched-chain amino acids. The ''ilvBNC'' operon encodes the ilvBN (ALS I) pair and a
ketol-acid reductoisomerase In enzymology, a ketol-acid reductoisomerase () is an enzyme that catalyzes the chemical reaction :(R)-2,3-dihydroxy-3-methylbutanoate + NADP+ \rightleftharpoons (S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH + H+ Thus, the two substrates of th ...
(ilvC). It is similarly regulated, but is specific to isoleucine and leucine; valine does not affect it directly. Both the ''ilvGMEDA'' and ''ilvBNC'' operons are derepressed during shortages of the branched-chain amino acids by the same mechanism that represses them. Both of these operons as well as the third, ''ilvIH'', are regulated by
leucine-responsive protein Leucine responsive protein, or Lrp, is a global regulator protein, meaning that it regulates the biosynthesis of leucine, as well as the other branched-chain amino acids, valine and isoleucine. In bacteria, it is encoded by the ''lrp'' gene. Lrp ...
(Lrp).


Inhibitors

Inhibitors of ALS are used as
herbicide Herbicides (, ), also commonly known as weedkillers, are substances used to control undesired plants, also known as weeds.EPA. February 201Pesticides Industry. Sales and Usage 2006 and 2007: Market Estimates. Summary in press releasMain page f ...
s that slowly starve affected plants of these
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
s, which eventually leads to inhibition of DNA synthesis. They affect grasses and dicots alike. They are not a chemistry class but rather mechanism class with diverse chemistries. The ALS inhibitor family includes
sulfonylurea Sulfonylureas (UK: sulphonylurea) are a class of organic compounds used in medicine and agriculture, for example as antidiabetic drugs widely used in the management of diabetes mellitus type 2. They act by increasing insulin release from the bet ...
s (SUs), imidazolinones, triazolopyrimidines (see :Triazolopyrimidines), pyrimidinyl oxybenzoates, and sulfonylamino carbonyl triazolinones. , the ALS inhibitors suffer the worst (known) resistance problem of all herbicide classes, having 169 known resistant target species. The structures of ALS herbicides are radically different from the normal substrate and so none of them bind at the
catalytic site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...
but instead at a site specific to herbicidal action. Therefore resistance mutations are expected to have widely varying effects on normal ALS catalysis activity, positive, negative and neutral. Unsurprisingly that is exactly what experiments have shown, including Yu ''et al.'', 2007 finding resistance in ''
Hordeum murinum ''Hordeum murinum'', commonly known as wall barley or false barley, is a species of grass. Overview ''Hordeum murinum'' is quite widespread and common. It flowers during May through July in mainly coastal areas. In the British Isles it is absent ...
'' due to a
proline Proline (symbol Pro or P) is an organic acid classed as a proteinogenic amino acid (used in the biosynthesis of proteins), although it does not contain the amino group but is rather a secondary amine. The secondary amine nitrogen is in the p ...
serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − for ...
substitution at amino acid 197 to ''increase'' ALS activity by 2x-3x.


Clinical significance

CADASIL CADASIL or CADASIL syndrome, involving cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, is the most common form of hereditary stroke disorder, and is thought to be caused by mutations of the '' Notch 3'' g ...
, an identified autosomal dominant condition characterized by the recurrence of subcortical infarcts leading to
dementia Dementia is a disorder which manifests as a set of related symptoms, which usually surfaces when the brain is damaged by injury or disease. The symptoms involve progressive impairments in memory, thinking, and behavior, which negatively affe ...
, was previously mapped to “ILVBL” gene within a 2-cM interval, D19S226–D19S199. This gene encodes a protein highly similar to the acetolactate synthase of other organisms. No recombination event was observed with D19S841, a highly polymorphic microsatellite marker isolated from a cosmid mapped to this region. No
mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, m ...
was detected on this gene in CADASIL patients, suggesting that it is not implicated in this disorder.


Interactions

In the study of
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Esc ...
, the FAD binding domain of ilvB has been shown to
interact Advocates for Informed Choice, doing business as, dba interACT or interACT Advocates for Intersex Youth, is a 501(c)(3) nonprofit organization using innovative strategies to advocate for the legal and human rights of children with intersex trai ...
with ilvN and activate the AHAS I enzyme.


References


External links

* *
Ramachandran plot In biochemistry, a Ramachandran plot (also known as a Rama plot, a Ramachandran diagram or a ,ψplot), originally developed in 1963 by G. N. Ramachandran, C. Ramakrishnan, and V. Sasisekharan, is a way to visualize energetically allowed regions ...
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