Pseudomonas active-site model

Two diketocamphane monooxygenases from Pseudomonas putida were found to catalyze the oxidation of a wide range of sulfides with modest enantioselectivities, and predictive active-site models were proposed [133],... 

Lemke, K. Lemke, M. Theil, F. A Three-dimensional predictive active site model for lipase from Pseudomonas cepacia. J. Org. Chem. 1997, 62, 6268. 

As increasing research has been carried out with these enzymes, a less empirical approach has been taken as a result of the different substrate profiles that have been compiled for various enzymes in this class. These profiles have been used to construct active site models for such versatile enzymes as the carboxylester hydrolase, pig liver esterase (PLE) (E.C. 3.1.1.1), and the microbial lipases (E.C. 3.1.1.3) from Burkholderia cepacia (formerly Pseudomonas cepacia) lipase (PCL), Candida... 

Theil, F., Femke, K., Ballschuh, S., Kunath, A., and Schick, H. 1995. Fipase-catalyzed resolution of 3-(aryloxy)-l,2-propanediol derivatives-towards an improved active-site model of Pseudomonas cepacia lipase (Amano PS). Tetrahedron Asymm., 6, 1323-1344. 

One of the reactions catalyzed by esterases and lipases is the reversible hydrolysis of esters (Figure 1 reaction 2). These enzymes also catalyze transesterifications and the asymmetrization of meso -substrates (Section 13.2.3.1.1). Many esterases and lipases are commercially available, making them easy to use for screening desired biotransformations without the need for culture collections and/or fermentation capabilities. As more and more research has been conducted with these enzymes, a less empirical approach is being taken due to the different substrate profiles amassed for various enzymes. These profiles have been used to construct active site models for such enzymes as pig liver esterase (PLE) (EC 3.1.1.1) and the microbial lipases (EC 3.1.1.3) Pseudomonas cepacia lipase (PCL), formerly P.fluorescens lipase, Candida rugosa lipase (CRL), formerly C. cylindracea lipase, lipase SAM-2 from Pseudomonas sp., and Rhizopus oryzae lipase (ROL) [108-116]. In addition, x-ray crystal structure information on PCL and CRL has been most helpful in predicting substrate activities and isomer preferences [117-119]. 

Bacterial Upases isolated from Pseudomonas fluorescens, P. aeruginosa, P. cepacia, and P. glumae are highly selective catalysts [442]. The stmctures of both of the latter enzymes were elucidated by X-ray analysis [443, 444], They seem to possess a narrower active site than CRL, since they are often unable to accommodate bulky substrates, but they can be extremely selective on slim counterparts [445 149]. Like the majority of the microbial lipases, the commercially available crude Pseudomonas sp. Upase preparations (PSL) all possess a stereochemical preference for the hydrolysis of the (/ )-esters of secondary alcohols, but the selectivity among the different preparations may differ to some extent [450], Various active-site models for PSL have been proposed [163, 451 53],... 

BFD from Pseudomonas putida has been characterized in detail with respect to its biochemical properties [4, 5] and 3D structure [6, 7]. Like other enzymes of this class, BFD is a homotetramer with a subunit size of about 56 kDa. The four active sites are formed at the interfaces of two subunits. The structure was published in 2003 [7] and contains the competitive inhibitor (R)-mandelate bound to the active sites, allowing model-based predictions about the interactions between active site residues and the substrate. 

As mentioned earlier (Section 4.2.1.1), empirical rules for the enantioselectivity of hydrolases have been developed. It is important to keep in mind that these rules do not work for all substrates. Most rules are based on pockets, which indicate how the steric bulk of the substituents in the substrate fit into the environment of the active site. Thus, such rules have been suggested for pig liver esterase(PLE) [66], the protease subtilisin [66-68], and certain lipases [69-71]. For secondary alcohols, most lipases follow the simple rule of Kazlauskas, which was developed for Pseudomonas cepacia, and which is depicted in Figure 4.4 [72]. This model implies that the fast-reacting enantiomers binds to the active site as described in Figure 4.4, whereas the slowly reacting one is not able to achieve a comfortable fit, because it will require the large substituent L to fit into the smaller pocket. In contrast to lipases, subtilisin displays opposite enantioselectivity toward secondary alcohols [68]. 

The ability of BVMOs to oxidize sulfur was also exploited by Beecher and Willetts in order to construct space filling cubic models of the active site of the DKCMO enzymes from Pseudomonas putida ATCC 17 453 (Fig. 16.5-40). They note that the more relaxed enantiospecificity of 36DKCMO, at least in terms of sulfoxidation, appears to be due to an overall larger 3D cubic space available in the active site11341. 36DKCMO appears to be the best candidate for a first X-ray structure of a BVMO, as preliminary crystal data have been reported1881. 

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