1. Molecular design of oxidoreductases for the biosynthesis of carbohydrate-based industrial polyols Research objectives: Our two objectives in this project are to develop industrial catalysts for the enzymatic productions of mannitol and sorbitol from glucose. Objective 1: Mannitol is produced enzymatically from fructose by mannitol dehydrogenase (MtDH) using NAD(P)H as the cofactor. It is theoretically possible to stoichiometrically convert glucose to mannitol in a single electrochemical reactor containing both immobilized thermostable MtDH and glucose isomerase. While thermostable glucose isomerases are commercially available, all known MtDHs are mesophilic enzymes. Our goal in this project was to clone a thermostable MtDH, characterize it, and engineer it for industrial application. Objective 2: Sorbitol can be produced from fructose by sorbitol dehydrogenase (SDH), but it can also be produced directly from glucose by aldose reductase (AR). Because no gene was identified in the genomes of hyperthermophiles that potentially encodes an SDH or an AR, our goals here were to express a fungal AR in Escherichia coli, characterize the enzyme’s catalytic and stability properties, and engineer this AR for high activity on glucose and high thermostability. Partial alignment of TM0298 with selected dehydrogenases. LrMtDH: L. reuteri MtDH (Genbank # AY090766); TmMtDH: T. maritima MtDH (Genbank # TM0298); HLADH: horse liver alcohol dehydrogenase (Genbank #P00328); TeSADH: Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (Genbank # U49975). Red: residues involved in catalytic Zn 2+ binding; blue: residues involved in structural Zn 2+ binding; green: consensus cofactor binding region. Of all protein sequences showing significant similarity to Leuconostoc mesenteroides MtDH (Genbank # AAM09029), a single one was from a hyperthermophile. Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase. When TM0298 was used as the query sequence for a BLASTp search of GenBank, though, the best scores against proteins of known function were against the Lactobacillus mesenteroides and L. reuteri MtDHs. TM0298 shares 31% identity and 52% similarity with these mesophilic MtDHs (see below). For this reason, we cloned TM0298 to characterize its substrate specificity. Effect of temperature on TmMtDH activity TmMtDH zinc content Objective 1: Characterization of a thermostable MtDH Research Objectives  Markers  Soluble crude extract  Heat-treated extract  TmMtDH after Ni-NTA column Expression and purification of TmMtDH TmMtDH     50 kDa 30 kDa 20 kDa The T. maritima mtdh gene was cloned in pET24a. From this construct, TmMtDH is expressed in Escherichia coli at high level with a C-terminal His-tag. TmMtDH has a specific activity of 85.2 unit/mg protein at 80°C on fructose with NADH as the cofactor (100% activity). It shows no detectable activity on glucose, xylose, threonine, acetaldehyde, or 2-butanone, but it shows 18% activity on D-xylulose, 29% on D-tagatose, and 5% on L-sorbose. In alcohol oxidation assays, TmMtDH is active on mannitol, but it shows no activity on sorbitol, xylitol, ethanol, or 2-butanol. TmMtDH properties TmMtDH is most active around 90°C Effect of pH on TmMtDH activity TmMtDH optimally reduces fructose at pH 5.5, and it optimally oxidizes mannitol at pH 8.5 (assays performed at 80°C) Effect of temperature on TmMtDH kinetic inactivation In 100 mM phosphate buffer (pH 7.0), TmMtDH has half-lives of 91 min at 75°C, 57 min at 80°C, 32 min at 85°C, 15 min at 90°C, and 6 min at 95°C. Zn 2+ in TmMtDH was titrated spectrophotometrically with ρ- hydroxymercuriphenyl sulfonate (PMPS) in the presence of 4-(2- pyridilazo)resorcinol. The ΔOD 500 of 0.43 reached at the plateau corresponds to 0.69 mol of Zn 2+ /subunit of enzyme. This result agrees with our atomic emission spectroscopy results that gave mol/mol TmMtDH. a Zn 2+ content of 0.73 Despite the fact that it contains four cysteines that could be involved in structural Zn 2+ binding, TmMtDH only contains a single, catalytic Zn 2+. Zn 2+, Mn 2+, and Co 2+ restore full activity to the EDTA-treated TmMtDH. ΔOD 250, ΔOD 500 TreatmentRelative activity (%) Control100 Plus 10 mM EDTA3.5 Plus 20 mM metals ZnCl 2 MnCl 2 CoCl 2 MgCl 2 CaCl 2 80.1 93.9 132.4 3.8 4.1 Production of mannitol from glucose in an electrochemical reactor combining TmMtDH with Thermotoga neapolitana xylose isomerase Electrode design First reactors run at 60°C, pH 6.0, with 300 mM glucose produce 130 mM mannitol in 5h. Why the reaction stops halfway is being investigated. Possible reasons include pH increase (up to 9.0). Objective 2: Expression of the Candida boidinii aldose reductase in Escherichia coli and enzyme characterization. The C. boidinii AR gene was cloned in pET24a. From this construct, AR is expressed in Escherichia coli at high level in soluble form, if induced at 30°C.  Crude extract  Soluble crude extract  After Q sepharose at pH 7.0  After Q sepharose at pH 8.3 ① ② ③ ④ AR 50 kDa 30 kDa C. Boidinii AR properties Publications in preparation: Hassler, B.L., Song, S.H., Vieille, C., Zeikus, J.G., and R.M. Worden. Coupling multiple enzymes to interfaces for bioelectronic applications. In preparation. Puttick, P., C. Vieille, S.H. Song, M.N. Fodje, P. Grochulski and L.T.J. Delbaere. Crystallization, preliminary X-ray diffraction and structure analysis of Thermotoga maritima mannitol dehydrogenase. Submitted to Acta Crystallog. Song, S.H., N. Ahluwalia, and C. Vieille. Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase. In preparation. V max (unit/mg protein) K m (mM) V max / K m Glucose7.90 ± 0.160.449 ± 0.02617.575 NADPH7.68 ± 0.230.05 ± 0.004153.6 NADHN.A. - Xylose38.7 ± 0.590.054 ± 0.004716.67 NADPH39.83 ± 0.640.031 ± 0.0021284.84 NADH0.5*0.18*2.78* Effect of pH on CbAR activity CbAR kinetic parameters at 37°C, pH 6.5 * Approximate values Further plans: Objective 1: We will use directed evolution to increase T. maritima MtDH’s activity on fructose at 60°C. We used T. maritima MtDH to develop a plate screening assay based on the oxidation of NADH by phenazine methosulfate, which in turn reduces nitroblue tetrazolium into an insoluble blue formazan dye. This assay can be used for any thermostable NAD(P)-dependent oxidoreductase. Objective 2. We will use directed evolution in combination with the screening assay developed with MtDH to engineer the C. boidinii AR into first a thermostable enzyme, and second a thermostable catalyst highly active on glucose. TmMtDH kinetic parameters at 80°C pH 6.0 * Approximate values Although TmMtDH is active with NADP(H), its affinity for NAD(H) is much higher, making it an NAD-dependent enzyme. pH 8.3 TmMtDH is the first thermostable mannitol dehydrogenase. Departments of Biochemistry & Molecular Biology 1 and Chemical Engineering 2 Michigan State University, East Lansing, MI 48824 Seung-Hoon Song 1, Brian Hassler 2, Mark Worden 2, J. Gregory Zeikus 1 (PD), and Claire Vieille 1 (co-PD) This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2005-35504-16239. * ** ***