Burkholderia cepacia lipase
Created by Anthony Kendle
The crystalline structure of a lipase-inhibitor complex was derived from X-ray diffraction of a protein in Burkholderia cepacia (formerly Pseudomonas cepacia) bacteria. As a lipase, B. cepacia lipase (BCL) plays a fundamental role in the degredation of lipids in bacteria biology. As a triacylgylcerol hydrolase, the main action of BCL is the hydrolysis of ester bonds that link glycerol to fatty acid chains1. The significance of BCL, among other lipases, is most readily observed in its role in commercial biosynthesis and biochemistry. As a true lipase, BCL is commercially useful in processing foods and oils. Studies of BCL with bound S-inhibitor offer new applications of BCL in industries such as pharmaceuticals2. Because of its enantiomeric preference for S-alcohols, BCL can be used in conjunction with a reaction kinetics assay in order to determine the absolute configuration of secondary alcohols3. This feature of BCL plays a crucial role in organic chemistry and pharmaceuticals in preparing enantiomerically pure samples of alcohols from racemic mixtures.
A homolog of BCL is derived from a different species Burkholderia glumae lipase (PGL) (PDB ID: 1QGE). PGL is a triacylgylcerol lipase that also binds a single calcium ion. Unlike BCL, PGL is a heterodimer consisting of a D-subunit (319 residues) and an E-subunit (97 residues)4. With a total of 319 amino acids in its quarternary structure and a molecular weight of 33174 Da, PGL has a deletion in its D-chain that corresponds to Pro-233 in BCL5. The isoelectric point of PGL is 5.95 (computationally derived using ExPASy software)6. BCL is homologous to PGL-D according to an E-value of 3e-136 and homologous to PGL-E according to an E-value of 3e-49[7]. The α/β-hydrolyase fold domain is present in both BCL and PGL, indicating conservation of the lipase function. In terms of structural homology, native BCL adopts an open conformation in comparison to the closed confirmation of PGL8. However, whenBCL is complexed with an S-inhibitor, its inactive, closed conformation more closely resembles the native structure of PGL.9
BCL consists of 320 amino acid residues that total a molecular weight of 33151 Da10. The isoelectric point is 5.42 (computationally derived using ExPASy software)6. The BCL-inhibitor complex studied by M. Luic et al is classified as a hydrolase complexed to a hydrolase inhibitor. The structural components and corresponding physiology of BCL is shown best through comparison of the crystalline structures of native BCL (PDB ID: 1OIL) and its form when bound by an S-inhibitor (PDB ID: 2NW6). The primary protein structure of BCL is defined by its sequence of 320 amino acid residues. Of this total sequence, 149 residues (46%) are hydrophobic (based on analysis of the primary sequence). Additionally, BCL contains cysteine residues at positions 190 and 270 that are capable of forming a disulfide bridge. The secondary structure of BCL sees the formation of α-helices intermixed with β-strands, however, the majority of the secondary structure of BCL is composed of α-helices1. BCL adopts its tertiary structure by folding into a globular protein. Once folded, BCL may adopt both open and closed conformations. No quaternary structure is observed for BCL, as it is formed from a single peptide chain and has no association with other subunits.
In addition to its amino acid constituents, BCL is able to bind prosthetic groups. Both native and inhibited crystalline structures of BCL show the binding of a single calcium ion at a calcium binding domain. This domain consists of four residues that engage in metal interactions with calcium. (Intermolecular interactions described below were determined using the RCSB PDB Ligand Explorer 3.9 program.)11 Ionic interactions occur between calcium and Asp242 and Asp288, such that the negative charge on the carboxylate moiety of the aspartate R-group stabilizes the positive charge (2+) on calcium: at cellular pH, these aspartate residues carry a negative charge. Additional interactions with calcium occur with Glu292 and Val296 via the partially negative oxygen of the carbonyl groups. BCL with S-inhibitor bound is also able to bind a single sodium ion through the carbonyl oxygen of Thr7. The crystalline structure of BCL without the bound inhibitor does not indicate binding of sodium.2
The prosthetic group introduced by M. Luic et al in determining the crystalline structure of inhibited BCL is the S-inhibitor itself: (1S)-1-(phenyoxymethyl)propyl methylphosphonochloridoate (POT).10 BLC binds POT by both hydrogen bonding and hydrophobic interactions. The hydrogen on the amide nitrogen of both Leu17 and Gln88 is a hydrogen bond donor to the oxygen of the phosphoryl oxygen of POT. The hydroxyl group of Ser87 also hydrogen bonds to this oxygen in addition to the oxygen of the phosphoester. The aromatic ring of POT participates in hydrophobic interactions via van der Waals forces through the R-groups of Leu17, Phe19, and Ala120. In addition to hydrophobic interactions contributed by Val266 and Ser87, the imidazole ring of His286 interacts with the chiral carbon on POT along with both carbons of the adjacent ethyl group.11
The key distinction between the two crystalline structures of BCL is due to a conformational change in the tertiary structure. The structure of the native BCL (1OIL) represents the open and active form. The enantiomerically inhibited BCL (2NW6) represents the closed and inactive form. The overall action of the conformational change is the result of cooperation ofmultiple protein domains. The mobile lid domain spans residues 118 to 150. The loop structure of this domain is composed of two α-helices connected by a β-turn. The positioning of polar and hydrophobic amino acids on this domain allow for a thermodynamically favorable conformational change. In the loop structure, polar amino acids are arranged on the outside (facing toward the solvent) while hydrophobic amino acids are arranged interiorly, facing the buried catalytic domain. At an aqueous-hydrophobic interface, the mobile lid lifts exposing a greater number of hydrophobic residues on the surface of BCL, which allows BCL to better interact with hydrophobic solvent while additionally exposing the catalytic site. This conformational change is further stabilized by interactions with Asp130. The conformational change at a solvent interface allows Asp130 of the lid to form hydrogen bonds with Thr132, Ser135, and Thr136[12].
The second significant domain of BCL is the β-hairpin that spans residues 214-228. Like the mobile lid, the β-hairpin domain is flexible. A highly hydrophobic domain, it is stabilized by the calcium ion that bound by BCL’s calcium binding domain. Experiments performed by Peter Trodler, Rolf D. Schmid, and Jurgen Pleiss showed that the movement of the β-hairpin at an oil-water interface is independent of the movement of the mobile lid12.
The catalytic domain of BCL is the α/β hydrolase fold12. The structure of this domain consists of parallel β-strands bordered by α-helicies. Three key residues within this domain define the catalytic site. Hydolysis of triacylglycerols is mediated by interactions with the catalytic triad of Ser87, His286, and Asp264. Because these catalytic residues do not occur in proximity in the amino acid sequence, it is evident that highly specific, conserved protein folding is present in BCL in order to bring residues of the primary structure into catalytic proximity. A similar catalytic triad is seen in generic serine proteases13. In this triad, the carboxy functional group of Asp264 accepts a hydrogen bond from the amine nitrogen of the imidazole ring of His286, stabilizing and orienting this residue. The imine nitrogen of the imidazole ring is able to abstract a proton from the hydroxyl functional group of Ser87. This increases the nucleophilicity of the serine residue, allowing it to attack ester linkages of triacylglycerol substrate to begin the process of hydrolysis. The presence of an S-inhibitor prevents the nucleophilic attack of Ser87. The hydroxyl group of Ser87 participates in a hydrogen bond with the oxygens of both the phosphoryl and phosphoester groups of POT, thus preventing deprotonation by His28611,14.
The inherent structure of BCL dictates its function. Specific, conserved folding of BCL gives rise to the association of remote primary structural elements to create specialized domains in the tertiary structure, such as the mobile lid and catalytic triad. As an interfacial protein, BCL’s structure allows for a coordinated conformational change in multiple domains upon contact with hydrophobic solvent that enables the lipase activity. The crystalline structures of both the open and closed conformations were analyzed through native BCL (PDB ID: 1OIL) and BCL with bound S-inhibitor (PDB ID: 2NW6). In both scenarios, the interaction of specific structural elements with the cellular environment results in a form-function complex that is vital to lipid metabolism.