Malate synthase

Malate synthase (EC 2.3.3.9) is an enzyme that catalyzes the chemical reaction

The three substrates of this enzyme are glyoxylic acid, acetyl-CoA, and water. Its products are the (S) enantiomer of malic acid, and coenzyme A. This enzyme participates in pyruvate metabolism and glyoxylate and dicarboxylate metabolism.

Nomenclature This enzyme belongs to the family of transferases, specifically acyltransferases that convert acyl groups into alkyl groups on transfer. The systematic name of this enzyme class is acetyl-CoA:glyoxylate C-acetyltransferase (thioester-hydrolysing, carboxymethyl-forming). Other names in common use include L-malate glyoxylate-lyase (CoA-acetylating), glyoxylate transacetylase, glyoxylate transacetase, glyoxylic transacetase, malate condensing enzyme, malate synthetase, malic synthetase, and malic-condensing enzyme.

Structure

Malate synthases fall into two major families, isoforms A and G. Isoform G is monomeric with a size of about 80-kD and found exclusively in bacteria. Isoform A is about 65 kD per subunit and can form homomultimers in eukaryotes. This enzyme contains a central TIM barrel sandwiched between an N-terminal alpha-helical clasp and an alpha/beta domain stemming from two insertions into the TIM barrel sequence. The enzyme terminates with a C-terminal five-helix plug. The active site, where the acetyl-CoA and glyoxylate bind to the enzyme, lies between the TIM barrel and C-terminal plug. Upon binding, the acetyl-CoA molecule forms a J-shape inserted into the binding pocket, by an intramolecular hydrogen bond between N7 of the adenine ring and a hydroxyl group on the pantetheine tail. In addition, a critical magnesium ion within the active site coordinates with glyoxylate, glutamic acid 427, aspartic acid 455, and two water molecules. The amino acids interacting with acetyl CoA upon binding are highly conserved. Sequence identity is high within each class of isoforms, but between both classes sequence identity drops to about 15%. The alpha/beta domain, which has no apparent function, is not seen in isoform A.

Mechanism The mechanism of malate synthase is an aldol reaction followed by thioester hydrolysis. Initially, aspartate 631 acts as a catalytic base, abstracting a proton from the alpha carbon of acetyl-CoA and creating an enolate that is stabilized by arginine 338. This is considered to be the rate-determining step of the mechanism. Then, the newly formed enolate acts as a nucleophile that attacks the aldehyde of glyoxylate, imparting a negative charge on the oxygen which is stabilized by arginine 338 and the coordinating magnesium cation. This malyl-CoA intermediate then undergoes hydrolysis at the acyl-CoA portion, generating a carboxylate anion. The enzyme finally releases malate and coenzyme A.

Function

The citric acid cycle (also known as the tricarboxylic acid cycle or the Krebs cycle) is used by aerobic organisms to produce energy via the oxidation of acetyl-CoA, which is derived from pyruvate (a product of glycolysis). The citric acid cycle accepts acetyl-CoA and metabolizes it to form carbon dioxide. A related cycle, called the glyoxylate cycle, is found in many bacteria and plants. In plants, the glyoxylate cycle takes place in glyoxysomes. In this cycle, isocitrate lyase and malate synthase skip over the decarboxylation steps of the citric acid cycle. In other words, malate synthase works together with isocitrate lyase in the glyoxylate cycle to bypass two oxidative steps of Krebs cycle and permit carbon incorporation from acetate or fatty acids in many microorganisms. Together, these two enzymes serve to produce succinate (which exits the cycle to be used for synthesis of sugars) and malate (which remains in the glyoxylate cycle). During this process, acetyl-CoA and water are used as substrates. As a result, the cell does not lose 2 molecules of carbon dioxide as it does in the Krebs cycle. The glyoxylate cycle, facilitated by malate synthase and isocitrate lyase, allows plants and bacteria to subsist on acetyl-CoA or other two carbon compounds. For example, Euglena gracilis, a single-celled eukaryotic alga, consumes ethanol to form acetyl-CoA and subsequently, carbohydrates. Within germinating plants, the glyoxylate cycle allows the conversion of reserve lipids into carbohydrates within glyoxysomes.

Evolutionary history Malate synthase is found as an octamer of identical subunits (each roughly 60kDa) in some plants, including maize. It is found as a homotetramer in the fungus Candida and as a homodimer in eubacteria. Malate synthase is fused to the C-terminus of isocitrate lyase in C. elegans, resulting in a single bifunctional protein. While there is currently not sufficient sequence information to determine the exact evolutionary history of malate synthase, plant, fungal, and C. elegans sequences are distinct and show no homologues from archaebacteria.

Activity in humans Traditionally, malate synthases are described in bacteria as part of the glyoxylate cycle, and malate synthase activity had not been reported for a human protein prior to a study by Strittmatter, et al. In this study, CLYBL was found to be a human mitochondrial enzyme with malate synthase activity. It is found in multiple eukaryotic taxa and is conserved in bacteria. CLYBL differs from other malate synthases in that it lacks a large portion of the C-terminal domain and shows lower specific activity and efficiency. CLYBL is linked to the vitamin B12 metabolism pathway because it is strongly co-expressed with MUT, MMAA, and MMAB, three members of the mitochondrial B12 pathway. Furthermore, a loss of function polymorphism, that leads to a loss of the CLYBL protein, is simultaneously associated with low levels of B12 in human plasma. While the exact mechanism of CLYBL’s involvement in B12 metabolism is not well understood, it is thought to convert citramalyl-CoA into pyruvate and acetyl-CoA. Without this conversion, itaconyl-CoA, a precursor to citramalyl-CoA, builds up in the cell leads to the inactivation of vitamin B12. This inactivation inhibits the methionine cycle, which leads to reduced serine, glycine, one-carbon, and folate metabolism.

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