Research On The Structure And Function Of A MaoC-Like Hydratase Involved In Biosynthesis Of Poiyhydroxyalkanoates

Phytophthora capsici is a worldwide soil borne disease. Pepper Phytophthora blight hasserious impact on pepper production all over the world, and it causes huge economic losses. Ithas a broad host range including pepper, pumpkin, cucumber, eggplant, tomato, and so on, upto more than20kinds of plants. In recent years there are many reports on its pathogenesis,pathogenic mechanism and control strategy, while there are few reports on searching for somebeneficial genes to serve mankind from such pathogens.In2008, the whole-genome draft sequence data from Photophthora capsici was madeavailable and updated constantly on line(http://genome.jgi-psf.org/Phyca11/Phyca11.home.html) by U.S. Department of Energy JointGenome Institute-DOE JGI. By bioinformatic analysis, more and more interesting genes arebeing found and this benefits us to reveal its function and be used by mankind. In our study,MaoC-like hydratase (to be shorten for MaoC), is one of such genes based on analysis of thegenome sequences. MaoC-like hydratase (MaoC), which belongs to (R)-hydratase involved inlinking the β-oxidation and the polyhydroxyalkanoates (PHAs) biosynthetic pathways, hasbeen identified recently.Problems concerning with the global environment have arisen much interest in thedevelopment of biodegradable polymers. Among the several biodegradable polymers underresearch, PHAs are good candidates for “green plastic” due to their biodegradable,biocompatible, thermoplastic, and mechanical properties. Because of this, considerableresearch has been undertaken to develop low cost and high efficiency processes for theproduction of PHAs. Reducing the cost of PHAs production may make PHA competitive as a“green plastic” alternative to conventional plastics which are difficult to degrade underrandom conditions. MaoC, possessing activity of (R)-hydratase involved in linking theβ-oxidation and the PHA biosynthetic pathways, has been recently identified in the fadBmutant Escherichia coli strain. MaoC is a new (R)-hydratase that catalyzes the (R)-specifichydration of the β-oxidation intermediate2-trans-enoyl-CoA to (R)-3-hydroxyacyl-CoA. ThusMaoC is an important enzyme in the biosynthesis of PHA because it supplies monomers thatare subsequently polymerized to form PHA by PHA synthase.We cloned a new MaoC gene from Photophthora capsici, and the crystal structure of theenzyme has been resolved at high resolution. The detailed function has been clarified on thebasis of the structure, showing as follows:1. We cloned a new beneficial gene named MaoC from pathogen Phytophthora capsici. MaoC was over-expressed in Escherichia coli and the recombinant MaoC was purified usingaffinity chromatography, anion-exchange chromatography and gel filtration chromatography.The purified recombinant MaoC was used to determine the Enoyl-CoA hydratase activityusing Crotonyl-CoA as the substrate, and the purified tagged MaoC showed the Enoyl-CoAhydratase activity of58.1U/mg towards Crotonyl-CoA. MaoC is a new member of(R)-hydratases which have been involved in biosynthesis of PHA, functioning as catalyzingthe (R)-specific hydration of the β-oxidation intermediate2-trans-enoyl-CoA to(R)-3-hydroxyacyl-CoA;2. MaoC was crystallized using the hanging drop vapour diffusion method, and thecrystal structure of the enzyme was resolved at1.93resolution by molecular replacement.The detailed description and analysis of three-dimensional structure were carried out: Thestructure shows that MaoC has a canonical (R)-hydratase folding with an N-domain and aC-domain, located between them is the intervening bridge region. The crystal structure of theenzyme belongs to the so-called “hot dog” fold superfamily, compared to the C-domain, theN-domain has an incomplete hot dog fold. MaoC forms a homodimer that is mediated by fourshort α-helices from each monomeric MaoC, and the residues participating in dimerization arehighly conserved among the (R)-hydratases. Further analysis of the structure reveals that thereis a hypothetical inhibitory segment in regulating the activity of MaoC.3. The active sites of MaoC (Asp-194, His-199and Gly-217) were identified by sequenceand structure alignment. To examine the importance of Asp-194, His-199and Gly-217forcatalysis, three mutants, D194N, H199Q, and G217A, were prepared and purified. To confirmthe presence and the apparent molecular mass of the purified mutant enzymes, circulardichroism, western blotting and gel filtration chromatography were carried out. The results ofactivity assay of the purified mutants show that Asp-194and His-199play a crucial role incatalysis, while Gly-217may be less important for catalysis. We propose that the threeresidues mentioned above play respective roles in the catalytic reaction of the enzyme asfollows. Asp-194may activate a water molecule by abstracting a proton from a watermolecule. The activated water molecule then attacks the carbon atom of crotonyl-CoA. Inaddition, His-199perhaps cooperatively donates a proton to the carbon atom of the substrate.Gly-217may hydrogen-bond to the carbonyl group of the thioester bond of the substrate.4. In its structure, MaoC forms a homodimer that is mediated by four short α-helices fromeach monomeric MaoC. Based on the sequence and structure alignment, the residuesparticipating in dimerization are highly conserved among the (R)-hydratases, and most of thecrystal structures of hydratases resolved thus far are dimers with a similar interface to that in MaoC. These structural observations suggest that the dimeric MaoC is important for itsfunction. Supporting this conclusion, three mutations (R23D, M27E and L191D) disruptingthe dimerization of MaoC resulted in monomeric MaoC proteins in solution as indicated by agel filtration assay. More importantly, the enzymatic activities were completely absent in theMaoC mutant proteins. Together, these data strongly support the notion that MaoC must bedimerized to function. Our study provides the first biochemical evidence for the functionalsignificance of the dimer. A crystal structure of the monomeric MaoC will provide significantinsight into the mechanism underlying the requirement of MaoC dimerization for itsenzymatic activity. Unfortunately, all the efforts to crystallize a monomeric MaoC proteinfailed.5. Based on the crystal structure, we made several mutants in engineering MaoC enzymeactivity in order to obtain mutants with higher activity compared to native MaoC. Structuralobservation suggests that a non-conserved insertion may have a role in regulating theenzymatic activity of MaoC. If this is the case, deletion of the insertion would generate aneffect on MaoC’s activity. To examine this possibility, we made single and deletion mutationssurrounding the hypothetical inhibitory segment and then assayed their effect on MaoC’sactivity. In full support of our prediction, one of the deletion mutant proteins, Del63-71,exhibited an activity of91.2U/mg in hydrolyzing enoyl-CoA, about1.5times that of the wildtype protein. Further deletion of the amino acids around the insertion (Del63-88) produced amore striking effect on promoting MaoC’s activity (126.7U/mg). In contrast, single mutationsin the insertion generated no significant effect on the activity of MaoC. The increased activityof the deletion mutant proteins was not caused by perturbation of the oligomerization status ofMaoC, because they both were eluted at a similar position to the wild type protein insize-exclusion chromatography. Collectively, our results indicate that an insertion region ofMaoC plays a role in inhibiting its activity.As the environmental problem is becoming more and more serious all over the world, it isobviously significant to develop the “green plastic” to improve the environment. Here wereport the crystal structure of a new enzyme involved in PHA biosynthesis. The data in ourstudy reveal the regulatory mechanism of an (R)-hydratase, providing information on enzymeengineering to produce low cost PHAs.