DESC:METHOD TO IDENTIFY ACTIVE SITE ON DECARBOXYLASE ENZYME

FIELD OF THE INVENTION
[0001] The present invention generally relates to the field of molecular biology and X-ray Crystallography. More specifically, it relates to a method to identify active site on decarboxylase enzyme and CO2 release pathway of the same.

BACKGROUND OF THE INVENTION
[0002] Decarboxylase catalyzes a spectrum of anabolic and catabolic pathways such as amino acid decarboxylation to carbohydrate synthesis, respectively. Currently, lot of studies are done on this class of enzyme and there are many poorly studied decarboxylases which are of commercial interest (6-methylsalicylic acid decarboxylase etc.).
[0003] ?-carboxymuconolactone decarboxylase (?-CMD) is a decarboxylase enzyme that belongs to the aromatic compound degradation pathway in bacteria. Although the first structure of ?-CMD (PDB: 2AF7) was solved in 2005 by the Northeast Structural Genomics Consortium, active site of the enzyme remains elusive. Detecting the active site residue of ?-CMD is quite challenging due to low sequence identity with any other well characterized enzymes. So is the case for the ?-CMD enzyme from catabolic pathway of Pseudomonas putida, called PcaC, of which the crystal structure is solved in the present invention. The enzyme contains 32% sequence identity with the first structure of ?-CMD (PDB: 2AF7).

SUMMARY OF THE INVENTION
[0004] The present invention solves the crystal structure of ?-carboxymuconolactone decarboxylase (?-CMD) from the catabolic pathway of Pseudomonas putida called PcaC. PcaC is a decarboxylase enzyme and its active site residues interacts with “carboxylic group”. Decarboxylases catalyzes the removal of carboxylic group from the substrate by producing CO2 as a byproduct. Bicarbonate closely mimic a carboxylic group as it is deprotonated form of carbonic acid (which is an example of a straight chained saturated carboxylic acid) and is a readily available substrate for soaking experiment.
[0005] In an embodiment of the present invention a method to identify active site in decarboxylase enzyme is disclosed. In an embodiment of the present method crystals of apo PcaC are soaked in an aqueous solution with variable soaking time and a number of data sets are collected based on the different soaking time. The datasets are refined against the Apo structure. Difference Fourier maps are analysed and a strong triangular positive difference electron density is located, and the density is unambiguously modelled as CO32-. In addition, elongated positive difference electron density is located in the vicinity of bound CO32-, which is modelled as CO2. The carbonate binding position helps to identify the active site residues. Different soaking time intervals helps to trace the action of the active site and the trailing CO2 molecules which reflects the product pathway.

OBJECT OF THE INVENTION
[0006] The object of the present invention is to provide a method to identify active site on decarboxylases enzyme and CO2 release pathway of the same.

BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Reference will be made to embodiments of the invention, examples of which may be illustrated in the accompanying figures. These figures are intended to be illustrative, not limiting. Although the invention is generally described in the context of these embodiments, it should be understood that it is not intended to limit the scope of the invention to these particular embodiments.
[0008] The objects, features, and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
[0009] Figure 1 illustrates active site after bicarbonate soaking.
[00010] Figure 2 illustrates comparison of the identified active site by the bicarbonate soaking method vs the autodocking method.
[00011] Figure 3 illustrates binding of the modelled bicarbonate to the active site of the orotidine 5'-phosphate decarboxylase correctly (PDB used: 3G18)

DETAILED DESCRIPTION
[0011] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are explanatory of the invention and are not intended to be restrictive thereof.
[0012] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” or “in an exemplary embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[00012] The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method.
[00013] ?-CMD is a decarboxylase enzyme that belongs to the aromatic compound degradation pathway in bacteria. Detecting the active site residue of ?-CMD is quite challenging due to low sequence identity with any other well characterized enzymes. The present invention solves the crystal structure of ?-CMD enzyme (Obtained from New York Structural Genomics Research Consortium, USA) from catabolic pathway of Pseudomonas putida called PcaC. PcaC is a decarboxylase enzyme and its active site residues interacts with “carboxylic group”. Decarboxylases catalyzes the removal of carboxylic group from the substrate by producing CO2 as a byproduct. Bicarbonate closely mimic a carboxylic group as it is a deprotonated form of carbonic acid (which is an example of a straight chained saturated carboxylic acid) and is a readily available substrate for soaking experiment.
[00014] In one of the embodiments of the present invention the crystals of apo PcaC are soaked in an aqueous solution (sodium bicarbonate) with variable soaking time and a number of data sets are collected based on the different soaking time. The datasets are refined against the Apo structure. Difference Fourier maps are analysed and a strong triangular positive difference electron density was located, and the density is unambiguously modelled as CO32-. In addition, elongated positive difference electron density is located in the vicinity of CO32-, which is modelled as CO2. The bicarbonate binding position helps to identify the active site residues. Different soaking time intervals helps to trace the action of the active site and the trailing CO2 molecules which reflects the product pathway. The basis of this method is that, the bicarbonate mimics the carboxylic group of the substrate and binds to the active site at the equivalent position of the substrate’s carboxylic group interacting with the exact active site residues of the PcaC enzyme. Furthermore, with increased soaking time of the crystals at room temperature, the bicarbonate sitting at the active site undergoes spontaneous dissociation to form the carbon dioxide which exits the enzyme via the protein’s product route, mirroring the actual decarboxylation reaction and its product path.
[00015] To confirm that the “bicarbonate soaking method” can identify the active site in other carboxylase enzymes, docking of the bicarbonate molecule in the native PcaC crystal structure was performed using AutoDock (i) to identify the location of active site. The docked carbonate at the active site was found to be identical to the one discovered by the soaking method as indicated by the superposition of the modelled PcaC-bicarbonate structure with the crystal structure of PcaC-bicarbonate complex (Figure 2). This shows that docking method can be used to evaluate success of bicarbonate soaking to identify the active sites in other carboxylase enzymes.
[00016] In the present invention, the bicarbonate molecule is modelled into the orotidine 5'-phosphate decarboxylase structure using AutoDock to check if bicarbonate moiety can correctly identify the active site in this decarboxylase as well. Orotidine 5'-phosphate decarboxylase is a well-studied decarboxylase whose active site residues are known. It has a catalytic triad comprising of D70, K72, and D75 (from the adjacent subunit) residues. In the orotidine 5'-phosphate decarboxylase-bicarbonate modelled structure, the bicarbonate binds at close proximity to the catalytic triad and is at hydrogen bonding distance with D70, K72, D75 and K42 (Figure 3). This proves that the bicarbonate soaking method can accurately identify active sites in other decarboxylases.
[00017] Biotin carboxylase is an important cellular carboxylase that uses a conserved biotin carboxylase component to catalyse the ATP-dependent carboxylation of biotin utilizing bicarbonate as the donor. Chou et al, 2009 had reported a 2 Å biotin carboxylase structure that were co-crystallized in the presence of Mg-ADP, biotin, and bicarbonate (PDB:3G8C). The resulting crystal structure contained bicarbonate molecule bound in the actives site, interacting with R292, V295 and E296. The orientation and binding of the bicarbonate with other substrates provided insight into the catalytic mechanism of biotin carboxylase. This is another case where the bicarbonate aids in the effective detection of the active site residues in decarboxylases by using crystallography. These above examples demonstrate the utility and scope of the bicarbonate soaking method in identification of active site in different decarboxylases enzymes.
[00018] Another embodiment of the present invention involves optimization of initial crystallization condition of apo PcaC and solving of the crytal structure using experimental phasing technique. Further, for bicarbonate soaking, a stock solution composed of 100 mM sodium bicarbonate and 40-60% glycerol is prepared seperately. The final sodium bicarbonate soaking solution prepared from the stock solution, is composed of 10 – 20 mM of sodium bicarbonate in presence of 20 – 25 %(v/v) ethylene glycol. (prepared using mother liquor). Further, the Apo PcaC crystals soaked for 1 minute, 5 minutes and 15 minutes in the sodium bicarbonate solution prior to cryo-cooling in the liquid nitrogen. X-ray diffraction data is collected from the frozen crystals. The Apo PcaC crystals are used for solving the apo-bicarbonate complexes using Molecular Replacement (MR) protocol of Auto-Rickshaw (an automated crystal structure determination platform). The resulting model for each complex, is refined in the program REFMAC5 and water molecules are added in difference map. The quality of the structure iteratively improved by refinement and model building. Finally, the highest peaks of the difference Fourier map are analysed specially those which did not look like density for water molecule. This way difference density for the ligands (CO32-and CO2) may be located using the graphic program Crystallographic Object-Oriented Toolkit (COOT). As shown in figure 1, the triangular positive difference electron density is built as CO32- whereas the elongated positive difference electron density was modelled as CO2.
[00019] The structural analysis from the soaked crystals (1 min) indicates that bicarbonate is bound at the active site along with two carbon dioxide molecules present at two different location near the active site, marking the product pathway (Figure 1). Soaking of crystal for 5 min shows binding of two CO2, one at the equivalent position of bicarbonate and another at the similar CO2 position. The longer soaked crystal (15 min) shows a clear site which is devoid of bicarbonate or carbon dioxide, demonstrating the complete conversion of bicarbonate into carbon dioxide and the subsequent diffusion of carbon dioxide from the active site.
[00020] Although the present disclosure has been described in the context of certain aspects and embodiments, it will be understood by those skilled in the art that the present disclosure extends beyond the specific embodiments to alternative embodiments and/or uses of the disclosure and obvious implementations and equivalents thereof. Thus, it is intended that the scope of the present disclosure described herein should not be limited by the disclosed aspects and embodiments above.
,CLAIMS:We Claim:
1. A method to identify active site in decarboxylase enzyme, the method comprising:
soaking apo PcaC crystals in an aqueous solution for a predetermined time;
collecting multiple data sets based on different soaking time;
refining the data sets against the apo structure;
analysing difference Fourier maps;
locating a strong triangular positive difference electron density;
modelling the density as CO32;
locating elongated positive difference electron density in the vicinity of CO32-; and
modelling said density as CO2.

2. The method as claimed in claim 1, wherein the aqueous solution comprises of sodium bicarbonate.
3. The method as claimed in claim 2, wherein the concentration of sodium bicarbonate is in the range 10 – 20 mM.
4. The method as claimed in claim 1, wherein the multiple data sets are collected at 5 minutes, 10 minutes and 15 minutes.
5. The method as claimed in claim 1, wherein the bicarbonate (CO32-) binding site helps in identifying the active site and the carbon dioxide (CO2) binding site maps the CO2 exit pathway in decarboxylase enzymes.