BACKGROUND: Genome-wide association studies (GWAS) have shown that variants in the 3-methylcrotonyl-CoA carboxylase (MCCC1)/lysosome-associated membrane protein 3 (LAMP3) loci (rs10513789, rs12637471, rs12493050) reduce the risk of Parkinson\'s disease (PD) in Caucasians, Chinese and Ashkenazi-Jews while the rs11248060 variant in the diacylglycerol kinase theta (DGKQ) gene increases the risk of PD in Caucasian and Han Chinese cohorts. However, their roles in Malays are unknown. Therefore, this study aims to investigate the association of these variants with the risk of PD in individuals of Malay ancestry.
METHODS: A total of 1114 subjects comprising of 536 PD patients and 578 healthy controls of Malay ancestry were recruited and genotyped using Taqman® allelic discrimination assays.
RESULTS: The G allele of rs10513789 (OR = 0.83, p = 0.001) and A allele of rs12637471 (OR = 0.79, p = 0.007) in the MCCC1/LAMP3 locus were associated with a protective effect against developing PD in the Malay population. A recessive model of penetrance showed a protective effect of the GG genotype for rs10513789 and the AA genotype for rs12637471. No association with PD was found with the other MCCC1/LAMP3 rs12493050 variant or with the DGKQ (rs11248060) variant. No significant associations were found between the four variants with the age at PD diagnosis.
CONCLUSIONS: MCCC1/LAMP3 variants rs10513789 and rs12637471 protect against PD in the Malay population.

Vitamin K-dependent carboxylation is a post-translational modification essential for the biological function of coagulation factors. Defects in carboxylation are mainly associated with bleeding disorders. With the discovery of new vitamin K-dependent proteins, the importance of carboxylation now encompasses vascular calcification, bone metabolism, and other important physiological processes. Our current knowledge of carboxylation, however, comes mainly from in vitro studies carried out under artificial conditions, which have a limited usefulness in understanding the carboxylation of vitamin K-dependent proteins in native conditions. Using a recently established mammalian cell-based assay, we studied the carboxylation of coagulation factors in a cellular environment. Our results show that the coagulation factor\'s propeptide controls substrate binding and product releasing during carboxylation, and the propeptide of factor IX appears to have the optimal affinity for efficient carboxylation. Additionally, non-conserved residues in the propeptide play an important role in carboxylation. A cell-based functional study of naturally occurring mutations in the propeptide successfully interpreted the clinical phenotype of warfarin\'s hypersensitivity during anticoagulation therapy in patients with these mutations. Unlike results obtained from in vitro studies, results from our cell-based study indicate that although the propeptide of osteocalcin cannot direct the carboxylation of the coagulation factor, it is required for the efficient carboxylation of osteocalcin. This suggests that the coagulation factors may have a different mechanism of carboxylation from osteocalcin. Together, results from this study provide insight into efficiently controlling one physiological process, such as coagulation without affecting the other, like bone metabolism.

Leptospira interrogans is the causative agent of leptospirosis. The in vitro growth of L. interrogans requires CO(2) and a partial 3-hydroxypropionate pathway involving two acyl-CoA carboxylases was suggested by genomic analysis to assimilate CO(2). Either set of the candidate genes heterologously co-expressed in Escherichia coli was able to demonstrate both acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) activities. The tri-subunit holoenzyme (LA_2736-LA_2735 and LA_3803), although failed to be purified, was designated ACC based on its substrate preference toward acetyl-CoA. The partially purified bi-subunit holoenzyme (LA_2432-LA_2433) has a considerably higher activity against propionyl-CoA as the substrate than that of acetyl-CoA, and thus, designated PCC. Native polyacrylamide gel electrophoresis indicated that this PCC has a molecular mass of around 669 kDa, suggesting an α(4)β(4) quaternary structure and both structural homology modeling and site-directed mutagenesis analysis of its carboxyltransferase subunit (LA_2433) indicated that the A431 residue located at the bottom of the putative substrate binding pocket may play an important role in substrate specificity determination. Both transcriptomic and proteomic data indicated that enzymes involved in the suggested partial 3-hydroxypropionate pathway were expressed in vivo in addition to ACC/PCC and the homologous genes in genomes of other Leptospira species were re-annotated accordingly. However, as the in vitro detected specific activity of ACC in the crude cell extract was too low to account for the growth of the bacterium in Ellinghausen-McCullough-Johnson-Harris minimal medium, further systematic analysis is required to unveil the mechanism of gluconeogenesis via anaplerotic CO(2) assimilation in Leptospira species.

In the reaction cycle of glutamate carboxylase, vitamin K epoxidation by O2 has been proposed to generate a very strong base able to remove a proton from the gamma carbon of a Glu residue, thus yielding a Glu-based carbanion that readily reacts with CO2. We have used hybrid density functional theory to study this appealing mechanism. Our calculations show a very exergonic four-step mechanism with the reaction of (triplet) O2 with the singlet vitamin K anion as the rate-limiting step, with a rate similar to the experimental value. Our study also establishes the need to apply continuum models when performing the optimization of minimum-energy crossing points between potential energy surfaces of different multiplicities for enzyme model systems.

Gamma-glutamyl carboxylase (GGCX) plays an important role in blood coagulation through post-translational carboxylation of vitamin K-dependent blood-clotting proteins. This carboxylation process is impaired in the presence of warfarin, a vitamin K antagonist. Recent studies on GGCX have provided insights into association of polymorphisms in this gene, with inter-individual differences in the required warfarin maintenance dose. In order to provide a useful resource for further elucidating this association, we here report a high-resolution single nucleotide polymorphism (SNP) and haplotype maps of an 18-kb genomic region corresponding to the GGCX locus in the Japanese population. Among 41 SNPs, seven insertion/deletion polymorphisms, and a microsatellite polymorphism that we detected by direct sequencing of the DNAs of 96 Japanese individuals who were treated with warfarin, 32 genetic variations have not been reported. Using genotype information from 12 SNPs and the EM algorithm, we estimated haplotypes for this genomic region. Subsequently, we investigated associations of each of these polymorphisms with the warfarin maintenance-dose requirements of 828 Japanese patients, including the 96 patients that were used for DNA sequencing. We found no significant association between the polymorphisms in GGCX and the dose requirement.

We studied proposed steps for the enzymatic formation of gamma-carboxyglutamic acid by density functional theory (DFT) quantum chemistry. Our results for one potentially feasible mechanism show that a vitamin K alkoxide intermediate can abstract a proton from glutamic acid at the gamma-carbon to form a carbanion and vitamin K epoxide. The hydrated carbanion can then react with CO2 to form gamma-carboxyglutamic acid. Computations at the B3LYP/6-311G** level were used to determine the intermediates and transition states for the overall process. The activation free energy for the gas-phase path is 22 kcal/mol, with the rate-limiting step for the reaction being the attack of the carbanion on CO2. Additional solvation studies, however, indicate that the formation of the carbanion step can be competitive with the CO2 attack step in high-dielectric systems. We relate these computations to the entire vitamin K cycle in the blood coagulation cascade, which is essential for viability of vertebrates.

A reaction path including transition states is generated for the Dowd mechanism [P. Dowd, R. Hershlne, S.W. Ham, S. Naganathan. Vitamin K and energy transduction: a base strength amplification mechanism. Science 269 (2005) 1684-1691] of action for Vitamin K carboxylase (VKC) using quantum chemical methods (B3LYP/6-311G**). VKC, an essential enzyme in mammalian systems, catalyzes the conversion of hydroquinone form of Vitamin K to the epoxide form in the presence of oxygen. An intermediate species of the oxidation of Vitamin K, an alkoxide, acts apparently to abstract the gamma hydrogen from specifically located glutamate residues. We are able to follow the Dowd proposed path to generate this alkoxide species. The geometries of the proposed model intermediates and transition states in the mechanism are energy optimized. We find that the most energetic step in the mechanism is the uni-deprotonation of the hydroquinone - once this occurs, there is only a small barrier of 3.5kcal/mol for the interaction of oxygen with the carbon to be attacked - and then the reaction proceeds downhill in free energy to form the critical alkoxide species. The results are consistent with the idea that the enzyme probably acts to facilitate the formation of the epoxide by reducing the energy required to deprotonate the hydroquinone form.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational modification of select glutamate residues of its vitamin K-dependent substrates to gamma-carboxyglutamate. In this report, we describe a new fluorescence assay that is sensitive and specific for the propeptide binding site of active carboxylase. We employed the assay to make three important observations: (1) A tight binding fluorescein-labeled consensus propeptide can be used to quantify the active fraction of the enzyme. (2) The off-rate for a fluorescein-labeled factor IX propeptide was 3000-fold slower than the rate of carboxylation, a difference that may explain how carboxylase can carry out multiple carboxylations of a substrate during the same binding event. (3) We show evidence that substrate binding to the active site modifies the propeptide binding site of carboxylase. The significant (9-fold) differences in off-rates for the propeptide in the presence and absence of its co-substrates may represent a release mechanism for macromolecular substrates from the enzyme. Additionally, sedimentation velocity and equilibrium experiments indicate a monomeric association of enzyme with propeptide. Furthermore, the carboxylase preparation is monodisperse in the buffer used for our studies.

gamma-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K-dependent posttranslational modification of glutamate to gamma-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

The conformational analysis of four glutamic acid analogues containing a cyclopentyl or cyclohexyl ring, substituted in position 1 by a Boc-protected amino group and a methyl ester group and in position 3 by a free carboxylate group (6-9), has been carried out in an aqueous environment, by 1H and 13C NMR spectroscopy, and molecular dynamics (MD). These compounds have been shown to be weak competitive inhibitors (Ki approximately 20-65 mM) of the vitamin K-dependent carboxylation of Boc-Glu-OMe in rat liver microsomes independently of their ring size and stereochemical features. However, the cyclic trans isomers have been found more active than the cis ones, and Boc-trans-C5-OMe (9) is the most potent inhibitor in the series (cis and trans isomers are defined by the relative arrangement of the carboxyl functions). Such cyclic glutamyl derivatives may provide valuable informations on the preferred bioactive conformations of synthetic glutamyl substrates at the active site of the carboxylase. In aqueous solution, the Boc-cis- and trans-C6 esters exhibit chair conformations with exclusively equatorial and axial substituent positions, while the Boc-cis- and -trans-C5 compounds may display envelope E or \'twist\' T conformations with the substituents in the following positions, equatorial; axial and isoclinal. For each compound, the conformations resulting from NMR and MD data were analyzed and classified according to the dihedral angles chi 1 and chi 2, the distances of functional groups, and the spatial charge distribution involving the free carboxyl group. A reduced number of conformational families were found to be in qualitative agreement with NMR and MD data. These results are discussed in relation with the carboxylase inhibitory activity of the analogues, and a spatial disposition of the glutamyl side chain that could be recognized by the carboxylase is deduced.