Chemistry & Biology

Previews present opportunities for drug discovery. The ability of enzymes such as EZH2 to reflect and amplify the underlying genomic causes of disease suggests chromatin modifiers will remain important therapeutic targets for the foreseeable future.

REFERENCES Bradley, W.D., Arora, S., Busby, J., Balasubramanian, S., Gehling, V.S., Nasveschuk, C.G., Vaswani, R.G., Yuan, C.-C., Hatton, C., Zhao, F., et al. (2014). Chem Biol. 21, this issue, 1463–1475. De Raedt, T., Beert, E., Pasmant, E., Luscan, A., Brems, H., Ortonne, N., Helin, K., Hornick, J.L., Mautner, V., Kehrer-Sawatzki, H., et al. (2014). Nature 514, 247–251.

Di Croce, L., and Helin, K. (2013). Nat. Struct. Mol. Biol. 20, 1147–1155. Garapaty-Rao, S., Nasveschuk, C., Gagnon, A., Chan, E.Y., Sandy, P., Busby, J., Balasubramanian, S., Campbell, R., Zhao, F., Bergeron, L., et al. (2013). Chem. Biol. 20, 1329–1339. Hidalgo, I., Herrera-Merchan, A., Ligos, J.M., Carramolino, L., Nun˜ez, J., Martinez, F., Dominguez, O., Torres, M., and Gonzalez, S. (2012). Cell Stem Cell 11, 649–662. Knutson, S.K., Wigle, T.J., Warholic, N.M., Sneeringer, C.J., Allain, C.J., Klaus, C.R., Sacks, J.D., Raimondi, A., Majer, C.R., Song, J., et al. (2012). Nat. Chem. Biol. 8, 890–896. Knutson, S.K., Warholic, N.M., Wigle, T.J., Klaus, Allain, C.J., Raimondi, A., Porter Scott, M., Chesworth, R., Moyer, M.P., Copeland, R.A., et al. (2013). Proc. Natl. Acad. Sci. USA 110, 7922–7927.

Martinez Molina, D., Jafari, R., Ignatushchenko, M., Seki, T., Larsson, E.A., Dan, C., Sreekumar, L., Cao, Y., and Nordlund, P. (2013). Science 341, 84–87. McCabe, M.T., Ott, H.M., Ganji, G., Korenchuk, S., Thompson, C., Van Aller, G.S., Liu, Y., Graves, A.P., Della Pietra, A., 3rd, Diaz, E., et al. (2012). Nature 492, 108–112. Qi, W., Chan, H., Teng, L., Li, L., Chuai, S., Zhang, R., Zeng, J., Li, M., Fan, H., Lin, Y., et al. (2012). Proc. Natl. Acad. Sci. USA 109, 21360–21365. Riising, E.M., Comet, I., Leblanc, B., Wu, X., Johansen, J.V., and Helin, K. (2014). Mol. Cell 55, 347–360. Shaknovich, R., and Melnick, A. (2011). Curr. Opin. Hematol. 18, 293–299.

Now Playing: Farnesol in the Biofilm Stephen A. Bell1 and Joseph Chappell1,* 1Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2014.11.001

The evolutionary pathway of specialized metabolism often takes unexpected, perplexing turns. In this issue of Chemistry & Biology, Feng and coworkers provide evidence for a unique phosphatase whose enzymatic product plays a critical role in biofilm formation in Bacillus subtilis. Microbial biofilms have intrigued the scientific community for centuries. Their peculiarities have been influential in prompting some of the earliest microscopic studies as well as the more modern, cutting edge molecular analyses (Høiby, 2014). Although not given full appreciation until the late 20th century, biofilms have forged an expanding scientific frontier that spans disciplines ranging from healthcare to agriculture (LappinScott et al., 2014). Their effects, good and bad, have continued to draw attention from researchers in many fields who hope to identify and/or exploit interesting and unique aspects of this ‘‘slimy’’ part of the microbial world. Despite the consensus that most microbes generate biofilms at some point during their lifecycle, considerable variations exist in the mechanisms through which biofilm formation is supported, even among the most extensively studied bacterial systems (Lo´pez et al., 2010).

One of these systems, the Gram-positive bacterium Bacillus subtilis, utilizes a common biofilm theme whereby a polysaccharide matrix infused with proteins anchors cells to one another and a surface (Vlamakis et al., 2013). While this and other areas of biofilm formation tend to follow common trends, other aspects of the process seem to be species specific, such as the signaling molecules that elicit the biofilm response (Lo´pez et al., 2010). In this issue of Chemistry & Biology, Feng et al. (2014) add a very interesting new layer to the requirements for biofilm assembly in B. subtilis. These authors provide evidence that not only does the B. subtilis squalene synthase-like enzyme (YisP) catalyze the formation of farnesol (FOH) from farnesyl diphosphate (FPP), but also that its product plays an important role in biofilm assembly. Earlier work by Lo´pez and Kolter (2010) originally proposed YisP to be a squalene synthase without truly identifying its reaction

product. Additionally, Lo´pez and Kolter (2010) demonstrated that when YisP was knocked out in B. subtilis, the ability to assemble biofilm was lost and therefore surmised that squalene played an important role in B. subtilis biofilm formation. However, close inspection of the protein sequence by Hu et al. (2013) during the elucidation of the crystal structure for YisP revealed that one of the canonical aspartate-rich motifs found in all squalene synthases was out of register in YisP. Thus, with the YisP crystal structures in hand (additional YisP structures were solved for this work) and the discrepancy found by Hu et al. (2013) in mind, the current authors calculated the volume of the YisP active site pocket and compared it to the active site pocket volume of the Staphylococcus aureus dehydrosqualene synthase (CrtM) and human squalene synthase (HsSS) enzymes. In comparing these measurements, Feng et al. (2014) realized that the YisP active site was likely

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Chemistry & Biology

Previews = Biofilm

= Farnesol = Species 1

= Bacillus subtilis

= Species 2

A

Surface

B

Surface Figure 1. Effect of FOH Produced by B. subtilis on Biofilm Formation in a Community of Mixed Species (A) When FOH is contained within B. subtilis cells, a biofilm community of mixed species is maintained. (B) As FOH from B. subtilis is released, the biofilm community is either broken apart or other species are unable to join.

too small to accommodate the two molecules of FPP needed for dehydrosqualene or squalene biosynthesis. This prompted them to test YisP with various prenyl diphosphate substrates (GPP, FPP, and GGPP) followed by chemical profiling of the reaction products. Surprisingly, the only product detected in these analyses was the dephosphorylated form of FPP, FOH. To confirm the role of the aspartate-rich motifs in YisP, site-directed mutants were constructed and tested. These results showed that the second aspartaterich motif was necessary for phosphatase activity. Restoration of the first aspartaterich motif in YisP had little effect on enzyme activity and did not give YisP the ability to catalyze a squalene synthaselike reaction. These efforts unequivocally demonstrated that YisP is an FPP phosphatase, not a squalene synthase. Reflecting back to earlier work by Lo´pez and Kolter (2010), where it was shown that the B. subtilis YisP deletion strain (DyisP) was unable to form biofilm, the authors of the current article asked what the role of FOH was in B. subtilis biofilm assembly. To address this, they supplied the wildtype and DyisP strains with exogenous FOH and observed no effect on wild-type biofilm assembly, whereas biofilm formation was restored in the mutant strain. The authors then delved deeper into the role of FOH in B. subtilis biofilm formation

and suggested that it acts to rigidify the membrane by altering the ‘‘phospholipid gel-to-liquid crystal phase transitions’’. Similar observations were made for staphyloxanthin and cholesterol, which are known to play important roles in membrane properties in S. aureus and eukaryotic cells, respectively. The current authors’ suggestion that FOH has a positive effect on biofilm formation in B. subtilis is intriguing when considered in the context of studies with Candida spp. (reviewed in Finkel and Mitchell, 2011) and Staphylococcus aureus (Jabra-Rizk et al., 2006; Kuroda et al., 2007). For example, it has been shown in Candida albicans that FOH inhibits filamentation, a critical part of this species’ ability to construct biofilm. In S. aureus, FOH has also been shown to affect biofilm formation negatively, although the effect was attributed to a less specific mechanism than that of C. albicans. Interesting to consider in this light is the effect FOH produced by B. subtilis might have in a biofilm community. Would the FOH produced by B. subtilis promote its own biofilm formation while inhibiting those of other species in a mixed biofilm community (Figure 1), thus giving B. subtilis a selective advantage? A final question arising from the current work is the evolutionary origin of YisP. Given its sequence and structural similar-

1422 Chemistry & Biology 21, November 20, 2014 ª2014 Elsevier Ltd All rights reserved

ities to CrtM and HsSS, where does YisP fall in the evolutionary pathway? The question here is whether these enzymes evolved from a common precursor, where one led to the other, or whether they co-evolved independently. Based on the conserved functional trait of CrtM, YisP and HsSS all being inhibited by zaragozic acid, one might assume that CrtM represents a primordial gene form that could have given rise to two independent evolutionary lines where one led to YisP and the other to eukaryotic squalene synthases, like HsSS. Certainly, other routes of evolution are possible. However, if we could piece together this evolutionary maze, then perhaps we would have another tool to help us unravel the molecular wizardry Mother Nature has used in capturing novel terpene biosynthetic capabilities as so elegantly demonstrated in the characterization of YisP by Feng et al. (2014).

ACKNOWLEDGMENTS The authors thank all the past and current J.C. laboratory members for their continued fascination with the chemistry and biochemistry of terpenes and the generous support provided by the NIH, NSF, USDA, and DOE to the J.C. laboratory.

REFERENCES Feng, X., Hu, Y., Zhgeng, Y., Zhu, W., Li, K., Huang, C.-H., Ko, T.-P., Ren, F., Chan, H.-C., Nega, M., et al. (2014). Chem Biol. 21, this issue, 1557–1563. Finkel, J.S., and Mitchell, A.P. (2011). Nat. Rev. Microbiol. 9, 109–118. Høiby, N. (2014). Pathog Dis 70, 205–211. Hu, Y., Jia, S., Ren, F., Huang, C.-H., Ko, T.-P., Mitchell, D.A., Guo, R.-T., and Zheng, Y. (2013). Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 69, 77–79. Jabra-Rizk, M.A., Meiller, T.F., James, C.E., and Shirtliff, M.E. (2006). Antimicrob. Agents Chemother. 50, 1463–1469. Kuroda, M., Nagasaki, S., Ito, R., and Ohta, T. (2007). FEMS Microbiol. Lett. 273, 28–34. Lappin-Scott, H., Burton, S., and Stoodley, P. (2014). Nat. Rev. Microbiol. 12, 781–787. Lo´pez, D., and Kolter, R. (2010). Genes Dev. 24, 1893–1902. Lo´pez, D., Vlamakis, H., and Kolter, R. (2010). Cold Spring Harb. Perspect. Biol. 2, a000398. Vlamakis, H., Chai, Y., Beauregard, P., Losick, R., and Kolter, R. (2013). Nat. Rev. Microbiol. 11, 157–168.

Now playing: farnesol in the biofilm.

The evolutionary pathway of specialized metabolism often takes unexpected, perplexing turns. In this issue of Chemistry & Biology, Feng and coworkers ...
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