In
enzymology, a maleate isomerase (
EC5.2.1.1), or maleate cis-tran isomerase, is a member of the Asp/Glu
racemase superfamily discovered in bacteria. It is responsible for catalyzing
cis-trans isomerization of the C2-C3 double bond in
maleate to produce
fumarate,[1] which is a critical intermediate in
citric acid cycle.[2] The presence of an exogenous
mercaptan is required for catalysis to happen.[3]
Analogous to other Asp/Glu racemase members, maleate isomerase is formed by two identical
protomers, with a flat dimerization surface.[13][14] Each protomer of maleate isomerase has two domains connected by a pseudo-twofold symmetry, with each domain contributes one catalytic cysteine, which is crucial to the isomerase activity at the active site.[5] Experiment shows that substitution of either cysteine by serine significantly reduces the rate of reaction of the enzyme.[1]
In addition to catalytic cysteines, a few other residues at the active site are important for the recognition of the substrate and help stabilize reaction intermediates.[5][1] For example, maleate isomerase from Pseudomonas putida S16 uses Asn17 and Asn169 form hydrogen bonds with the carboxylate group of the maleate distal to Cys82.[5] Tyr139 hydrogen bonds with the carboxylate group of the maleate proximal to Cys82.[5] Pro14 and Val84 make van der Waals interactions with the C2 and C3 carbon atoms of the maleate.[5]
Mechanism
The mechanism of maleate isomerase is considered to be similar to other Asp/Glu racemase members, though have not been fully understood. One proposed reaction mechanism of Nocardia farcinia maleate isomerase is as follows.[1][9] At the active site of maleate isomerase, Cys76 is first deprotonated to be more readily act as a nucleophile.[1] The sulfur atom of the deprotonated Cys76 then carries a direct nucleophilic attack to the C2 atom of the maleate, covalently bonding to the C2 atom.[9][1] Concomitantly, thiol proton of Cys194 is transferred onto the C3 atom of the maleate to form a succinyl-cysteine intermediate.[9][1] The newly formed C2–C3 single bond is then rotated, with Cys76S–C2 bond dissociated, and C3 atom of the maleate deprotonated by Cys194, thus forming fumarate with regeneration of a neutral Cys194.[9][1] In certain type of bacteria, maleate seems completely buried inside the cavity of maleate isomerase and cannot be seen on the surface of the enzyme.[5]
Industrial relevance
Maleate isomerase can be used to produce fumaric acid, an important building block material for
polymerization and
esterification reactions, from the isomerization of maleic acid.[7] Maleic acid is produced from
maleic anhydride.[7]
Maleic acid can also be converted into fumaric acid by thermal or catalytic cis–trans isomerization.[15][16] However, these conversion methods are occurring at high temperatures that causes formation of by-products from maleic and fumaric acids, as a result, yields are below the equilibrium yields.[17] This problem was the main motivation for the alternative enzymatic strategy with maleate isomerase that would facilitate isomerization without by-products.[7]
It is known that, even at moderate temperatures, natural maleate isomerase is unstable.[18] For that reason, heat-stable maleate isomerases are engineered and applied.[7] For example, thermo-stable maleate isomerases derived from Bacillus stearothermophilus,
Bacillus brevis, and
Bacillus sporothermodurans were used to improve the process.[7][17] In a study using Pseudomonas alcaligenes XD-1, conversion rate from maleic acid into fumaric acid could be achieved as high as 95%.[19][20][7]
References
^
abcdefghiFisch F, Fleites CM, Delenne M, Baudendistel N, Hauer B, Turkenburg JP, Hart S, Bruce NC, Grogan G (August 2010). "A covalent succinylcysteine-like intermediate in the enzyme-catalyzed transformation of maleate to fumarate by maleate isomerase". Journal of the American Chemical Society. 132 (33): 11455–7.
doi:
10.1021/ja1053576.
PMID20677745.
^
abHatakeyama K, Goto M, Kobayashi M, Terasawa M, Yukawa H (July 2000). "Analysis of oxidation sensitivity of maleate cis-trans isomerase from Serratia marcescens". Bioscience, Biotechnology, and Biochemistry. 64 (7): 1477–85.
doi:
10.1271/bbb.64.1477.
PMID10945267.
^
abcdeDokainish HM, Ion BF, Gauld JW (June 2014). "Computational investigations on the catalytic mechanism of maleate isomerase: the role of the active site cysteine residues". Physical Chemistry Chemical Physics. 16 (24): 12462–74.
doi:
10.1039/c4cp01342e.
PMID24827730.
^Hatakeyama K, Asai Y, Uchida Y, Kobayashi M, Terasawa M, Yukawa H (October 1997). "Gene cloning and characterization of maleate cis-trans isomerase from Alcaligenes faecalis". Biochemical and Biophysical Research Communications. 239 (1): 74–9.
doi:
10.1006/bbrc.1997.7430.
PMID9345272.
^Hatakeyama K, Goto M, Uchida Y, Kobayashi M, Terasawa M, Yukawa H (March 2000). "Molecular analysis of maleate cis-trans isomerase from thermophilic bacteria". Bioscience, Biotechnology, and Biochemistry. 64 (3): 569–76.
doi:
10.1271/bbb.64.569.
PMID10803955.
S2CID43798064.
^Ohtaki A, Nakano Y, Iizuka R, Arakawa T, Yamada K, Odaka M, Yohda M (March 2008). "Structure of aspartate racemase complexed with a dual substrate analogue, citric acid, and implications for the reaction mechanism". Proteins. 70 (4): 1167–74.
doi:
10.1002/prot.21528.
PMID17847084.
S2CID38854552.
^Takamura Y, Takamura T, Soejima M, Uemura T (January 1969). "Studies on the Induced Synthesis of Maleate Cis-Trans Isomerase by Malonate: Part III. Purification and Properties of Maleate cis-trans Isomerase Induced by Malonate". Journal Agricultural and Biological Chemistry. 33 (5): 718–728.
doi:
10.1080/00021369.1969.10859369.
^Nakajima-Kambe, Toshiaki; Nozue, Takehiro; Mukouyama, Masaharu; Nakahara, Tadaatsu (January 1997). "Bioconversion of maleic acid to fumaric acid by Pseudomonas alcaligenes strain XD-1". Journal of Fermentation and Bioengineering. 84 (2): 165–168.
doi:
10.1016/S0922-338X(97)82549-4.
^Ichikawa, Sosaku; Iino, Tomoko; Sato, Seigo; Nakahara, Tadaatsu; Mukataka, Sukekuni (January 2003). "Improvement of production rate and yield of fumaric acid from maleic acid by heat treatment of Pseudomonas alcaligenes strain XD-1". Biochemical Engineering Journal. 13 (1): 7–13.
doi:
10.1016/S1369-703X(02)00080-3.