A Tight Fit The structure of a mutant form of SbADC (Y252F) with the product of the reaction with 4-nitro-cinnamaldehyde bound shows that the bound product fills most of the available space in the active site.

Meet SbADC, The Secret Ingredient to Green Chemistry

Saint Joan Antida SMART Team: Velari Araujo, Lissette Delatorre, Alexis Lockett-Glover, Erika Johnson, Darneisha Virginia, Ashley Volmer, and Fatima Yacoob.

Advisor: Cindy McLinn Mentor: Nicholas Silvaggi, Ph.D., University of Wisconsin – Milwaukee

Introduction Science is continuously improving the world we live in, however each advance comes with new problems. Eco-friendly processes are always needed. Green chemistry is a growing field that looks for new ways to synthesize chemicals that use less energy and/or less and safer solvents. People (most) realize that we have one earth and one body and they both need to be taken care of. With green chemistry we can improve and coexist in a world in an innovative way.

Abstract Scientists continue to seek eco-friendly methods and materials to be used in chemical synthesis, since there are many harmful solvents such as benzene currently in use. The use of certain enzymes could make syntheses “greener” because they work in water at room temperature and near neutral pH. However finding an enzyme that does exactly what you need it to is very difficult, so it is important that scientists try to identify new enzymes with potentially useful catalytic activities. Streptomyces bingchenggensis acetoacetate decarboxylase (SbADC) may be useful in some green chemistry applications. The enzyme SbADC comes from the same family as acetoacetate decarboxylase (ADC) which cleaves acetoacetate into the products carbon dioxide and acetone. While it is not known what SbADC does in a living bacterium researchers know what SBADC can do in a test tube. SbADC functions as an aldolase-dehydratase, forming a double-bond between pyruvate and a range of aldehydes. SbADC has been shown to bind to the substrate 4-nitro-cinnamylidenepyruvate. Our mentor would like to know what other analogs of this compound might bind with SbADC. Specifically, he is interested to know if the enzyme will accept substrates with groups on the ortho- and meta-positions of the cinnamyl ring. Since these “green” compounds are not available and are difficult to make, the St. Joan Antida SMART (Students Modeling a Research Topic) Team has used 3-D printing technology to create models of the protein and a selection of potential substrates in order to learn more about the substrate specificity of SbADC.

References: Silvaggi, N., et.al (2012), [SbADC Kinetics Study], Unpublished data. V. Andrianov et al., Eur. J. Med. Chem. 44 (2009) 1067-1085

Surface views of SbADC. The outer surface of the protein is silver, while the inner surface is semi-transparent black.

Conclusion SbADC is the first member of the Acetoacetate Decarboxylase Superfamily (other than the original acetoacetate decarboxylase) to have its structure and catalytic mechanism studied. Instead of being a decarboxylase, SbADC actually takes two pieces – pyruvate and an aldehyde – and forms of double bond between them. Since reactions that form carbon-carbon bonds can be tricky for synthetic organic chemistry, SbADC may be a useful tool in “green chemistry” applications. As we learn more about how SbADC and related enzymes work, it may be possible to engineer this enzyme – change specific side chains in the active site – to make SbADC catalyze a slightly different reaction or use a different aldehyde as a substrate.

Unpublished data The Problem

Synthetic chemistry is the science of building complex new molecules by reacting simpler building blocks. For these reactions to take place, the building blocks need to be dissolved in a solvent. Many of the chemicals used in chemical synthesis are not soluble in water, so potentially hazardous organic solvents must be used in many cases. It is also common for reactions to be done at very high or very low temperatures, which can consume large amounts of energy. When reactions are complete the solvent has to be disposed of, which is expensive. Also, the use of hazardous solvents poses risks to workers and the environment.

Enzymes in Green Chemistry Enzymes play a valuable role in green chemistry, because they are able to catalyze chemical reactions in water at room temperature and neutral pH. However, their extreme reaction and substrate specificity make it difficult or impossible to find an enzyme that will do what you want it to. So, it is important to find and study new enzymes that catalyze reactions that might be useful in green chemistry. In a way, it is like finding new tools for the green chemistry “toolbox.” The acetoacetate decarboxylase superfamily (ADCSF) is a large and unexplored group of enzymes. ADCSF enzyme from Streptomyces bingchenggenis (SbADC) is an aldolase-dehydratse that can condense- form chemical bonds between- pyruvate and a number a different aldehydes.

SbADC is a New Kind of Aldolase SbADC forms new carbon-carbon bonds between a donor substrate, pyruvate, and a whole range of acceptor substrates (various unsaturated aldehydes). A proposed mechanism for the SbADC-catalyzed reaction is shown below (left), where E84 acts as an acid-base catalyst.

Carbon-carbon bond formation can be tricky using traditional synthetic chemistry. SbADC might be a useful tool for green chemistry, because it creates C-C bonds between pyruvate and a surprising range of aldehyde substrates. The steady-state kinetics data (see table, below right) show that, while SbADC will accept short aliphatic aldehydes as substrates, the preferred substrate is a substituted cinnamaldehyde. The goal of this modeling study was to figure out which positions on the cinnamyl ring could be substituted and which could not.

The Structure of SbADC SbADC is active as a homo-tetramer (28 kDa per protomer, below left). Our 3-dimensional model of chain C (below right), showing residues K122 and Y252 at the catalytic center.

The model of the protein, together with the substrate “action figures” that can be magnetically docked into the protein, clearly show that only substrates with groups in the para position are likely to be good substrates for SbADC.

Model based on SbADC.pdb (unpublished data)

A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973).