Abstract Enzymes catalyze chemical changes in biological systems. Therefore, to understand the chemistry of living systems, it is important to understand the enzyme structure and the chemistry of the enzyme's functional groups which are involved in catalysis. In this study, structure and function relationships of two enzymes, (1) α-methylacyl-CoA racemase from Mycobacterium tuberculosis (MCR) and (2) argininosuccinate lyase from Escherichia coli (eASL) have been studied using X-ray crystallography. The main focus of this study has been understanding the structure-function relationship of MCR. The eASL has been crystallized from a highly concentrated sample of purified recombinant α-methylacyl-CoA racemase in which it occurred as a minor impurity. The structure of eASL has been solved using molecular replacement at 2.44 Å resolution. The enzyme is a tetramer, but in this crystal form there is a dimer in the asymmetric unit. Each active site is constructed from loops of three different subunits. One of these catalytic loops, near residue Ser277 and Ser278, has been disordered in the previous structures of active lyases, but is very well ordered in this structure in one of the subunits due to the presence of two phosphate ions in the respective active site cavity. The positions of these phosphate ions indicate a plausible mode of binding of the succinate moiety of the substrate in the competent catalytic complex and therefore this structure has provided new information on the reaction mechanism of this class of enzymes. α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of α-methyl-branched CoA esters. An Amacr homologue from the eubacteria Mycobacterium tuberculosis, referred to as MCR, was taken as a model protein. MCR was purified, crystallized and the structure of unliganded protein was determined at 1.8 Å resolution using the MIRAS procedure. The structure shows that the enzyme is an interlocked dimer. To understand the reaction mechanism and the mode of substrate binding, several crystallographic binding studies were done using both wild type MCR and mutant H126A MCR crystals. In particular, the structures of the wild type MCR-complexes with (R, S)-ibuprofenoyl-CoA (1.85 Å), (R)-2-methylmyristoyl-CoA (1.6 Å) and (S)-2-methylmyristoyl-CoA (1.7 Å) were important in this respect. These crystal structures show that Asp156 and His126 are the two catalytic residues which are involved in proton donation and abstraction, respectively; when the (S)-enantiomeric substrate is bound in the active site and vice versa when the (R)-enantiomeric substrate is bound. The tight geometry of the active site also shows that His126 and Asp156 are involved in stabilizing the transition state. These crystal structures show that in the active site of MCR, there is one binding pocket for the CoA part and there are two different binding pockets (R-pocket and S-pocket) connected by a hydrophobic methionine rich surface for binding the fatty acyl part of the substrate. After substrate binding, proton abstraction takes place which produces a planar intermediate. Then, donation of a proton to the other side of the planar intermediate changes the configuration at the chiral center. During the stereochemical interconversion of the two enantiomers, the acyl group moves between R-pocket and S-pocket by sliding over the hydrophobic surface connecting these two pockets.

Abstract α-Methylacyl-CoA racemase (Amacr) is an enzyme at the merging point of two important pathways of lipid metabolism: elimination of methyl-branched fatty acids and synthesis of bile acids. Amacr is regarded as obligatory for these processes. Patients with Amacr-deficiency suffer from adult onset sensory motor neuropathy and/or severe neonatal cholestasis with coagulopathy and fat-soluble vitamin malabsorption. Amacr is also linked to cancer and so far has been proposed as a new marker for diagnosis of at least prostate and colon cancers. Common sources of phytol derived branched-chain fatty acids for man are ruminant fats, meat and dairy products. The bile acid synthesis is the main pathway for cholesterol catabolism. Amacr is considered to be a member of family III of the CoA transferases (L-carnitine dehydratase - bile acid inducible protein F (CaiB-BaiF) family) and localized to two subcellular compartments, mitochondria and peroxisomes. In this work the mouse gene encoding Amacr was characterized, the gene was inactivated and mutational and structural studies were used to determine the loop and the active site structure of the enzyme. It was shown that mouse Amacr which locates both to mitochondria and peroxisomes, is an identical product of a single gene, which is located at chromosome 15, region 15B1. Neither alternative replication, splicing, or any post-translational modifications of the enzyme occur. The mouse model for Amacr-deficiency indicated a role of Amacr in detoxification of methyl-branched fatty acids, and suggested that a diet free from these phytol metabolites may function as a treatment for the deficiency. Furthermore, major changes were observed in the bile acid pool of the knock-out mice compared to wild type mice. However, the study suggests that there is an Amacr-independent pathway for synthesis of bile acids albeit of low capacity, which provides a way for Amacr-deficient individuals to survive. The mutational and structural studies confirmed Amacr as a member of family III of the CoA transferases. Furthermore, according to comparisons of the structural data of Amacr and other members of the family (FRC, YfdW), the superfamily can be divided into two subgroups, racemases and transferases. Proteins in the subfamilies share the CoA-binding mode, but the substrate specificities as well as the catalysed reaction differ greatly.

Abstract α-Methylacyl-CoA racemase (Amacr) is an auxiliary enzyme of β-oxidation and participates in the elimination of methyl-branched fatty acids in peroxisomes and in mitochondria and in the synthesis of bile acids in peroxisomes. Amacr catalyzes in reversible manner the isomerization of fatty acyl-CoA esters with a methyl group in the R-configuration to the corresponding S-configuration, which allows them to serve as substrates for the next reaction in their metabolism. The substrates of Amacr include the acyl-CoA esters of 2R-pristanic acid, a metabolite derived from phytol, and 25R-THCA and 25R-DHCA (tri- and dihydroxycholestanoic acid), the bile acid intermediates derived from cholesterol. AMACR-deficiency in humans results in the accumulation of R-isoforms of its substrates. Patients with adult onset AMACR-deficiency suffer from neurological disorders. The more severe infantile form of the deficiency is characterized by liver disease. Amacr-deficient mice show a bile acid pattern similar to that of human patients with accumulation of bile acid intermediates in their body. In contrast to humans, Amacr-deficient mice are clinically symptomless on a regular laboratory chow diet. Supplementation of phytol in their diet triggers the disease state with liver abnormalities. In this study it was shown that in spite of the disruption of a major metabolic pathway, Amacr-deficient mice are able to readjust their cholesterol and bile acid metabolism to a new balanced level allowing them to live a normal life span. A double knockout mouse model deficient in Amacr and MFE-1 (peroxisomal multifunctional enzyme type 1) was generated in this work. Characterization of this mouse line showed that MFE-1 can contribute to peroxisomal side-chain shortening of C27 bile acid intermediates in both Amacr-dependent and Amacr-independent pathways. In addition, this work confirmed that Amacr-deficient mice are unable to thrive when phytol is supplemented in their chow. The main cause of death was liver failure accompanied by kidney and brain abnormalities. The detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways and Amacr-deficient mice are not able to respond adequately. The results of this study emphasize the indispensable role of Amacr in detoxification of α-methyl branched fatty acids. / Tiivistelmä α-Metyyliasyyli-koentsyymi-A-rasemaasi (Amacr) osallistuu metyyli-haarautuvien rasvahappojen eliminointiin peroksisomeissa ja mitokondrioissa ja sappihappojen synteesiin kolesterolista peroksisomeissa. Amacr katalysoi käänteisesti rasvahappojen asyyli-koentsyymi-A-estereiden isomerisaatio-reaktiota, jossa stereokemiallisesti R-asemassa oleva metyyliryhmä siirtyy S-asemaan. Tämä on edellytys eliminointiketjun seuraavan reaktion tapahtumiselle. Amacr-entsyymin substraatteja ovat fytolin aineenvaihdunnassa syntyvän 2R-pristaanihapon ja kolesterolista sappihapposynteesireitin välituotteina syntyvien 25R-trihydroksikolestaanihapon ja 25R-dihydroksikolestaanihapon (25R-THCA ja 25R-DHCA) asyyli-koentsyymi-A-esterit. Ihmisellä Amacr-entsyymin puutos johtaa R-muodossa olevien substraattien kertymiseen, joka aiheuttaa neurologisia oireita aikuisiässä alkavassa sairauden muodossa. Lapsuusiässä alkavassa tautimuodossa potilaille kehittyy vakava maksasairaus. Tutkimuksen tulokset osoittivat, että Amacr-poistogeenisten hiirten elinikä ei lyhene huolimatta yhden tärkeän aineenvaihduntareitin estymisestä. Tämä on hyvä esimerkki siitä, kuinka nisäkäs pystyy mukauttamaan kolesteroli- ja sappihappoaineenvaihduntaansa vastaamaan muuttunutta tilannetta aineenvaihdunnassa. Tässä työssä tuotettiin myös kaksoispoistogeeninen hiirimalli, jonka Amacr- ja peroksisomaalinen monitoiminnallinen entsyymi tyyppi 1- (MFE-1) entsyymit ovat toimimattomat. Tämä hiirimalli paljasti, että MFE-1 pystyy osallistumaan 27:ää hiiltä sisältävien sappihappovälituotteiden sivuketjun lyhentämiseen sekä Amacr entsyymin kanssa että ilman sitä. Työn tulokset myös osoittivat, että Amacr-poistogeeniset hiiret eivät ole elinkykyisiä, jos niiden ravinto sisältää fytolia. Maksan toiminnanvajaus oli näiden hiirten tärkein kuolinsyy, mutta hiirten munuaisten ja aivojen kudosrakenteissa oli myös muutoksia. Maksassa fytolin metaboliittien vaarattomaksi tekeminen aiheuttaa villityypin hiirillä useamman aineenvaihduntareitin aktivoitumisen, mutta Amacr-poistogeeniset hiiret eivät pysty reagoimaan tähän samalla tavalla. Tämä työ osoittaa, että Amacr-entsyymin elintärkeä tehtävä on osallistua ravinnon mukana elimistöön joutuvien α-metyylihaarautuvien rasvahappojen eliminaatioon.