Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.     

Xylene monooxygenase catalyzes the multistep oxygenation of toluene and pseudocumene to corresponding alcohols, aldehydes, and acids in Escherichia coli JM101

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes the methylgroup hydroxylation of toluene and xylenes. To investigate the potential of xylene monooxygenase to catalyze multistep oxidations of one methyl group, we tested recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA under the control of the alk regulatory system of Pseudomonas oleovorans Gpo1. Expression of xylene monooxygenase genes could efficiently be controlled by n-octane and dicyclopropylketone. Xylene monooxygenase was found to catalyze the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes. For all three transformations (18)O incorporation provided stong evidence for a monooxygenation type of reaction, with gem-diols as the most likely reaction intermediates during the oxygenation of benzyl alcohols to benzaldehydes. To investigate the role of benzyl alcohol dehydrogenase (XylB) in the formation of benzaldehydes, xylB was cloned behind and expressed in concert with xylMA. In comparison to E. coli expressing only xylMA, the presence of xylB lowered product formation rates and resulted in back formation of benzyl alcohol from benzaldehyde. In P. putida mt-2 XylB may prevent the formation of high concentrations of the particularly reactive benzaldehydes. In the case of high fluxes through the degradation pathways and low aldehyde concentrations, XylB may contribute to benzaldehyde formation via the energetically favorable dehydrogenation of benzyl alcohols. The results presented here characterize XylMA as an enzyme able to catalyze the multistep oxygenation of toluenes.     

Use of the two-liquid phase concept to exploit kinetically controlled multistep biocatalysis

The two-liquid phase concept was used to develop a whole cell biocatalytic system for the efficient multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde. Recombinant Escherichia coli cells were employed to express the Pseudomonas putida genes encoding xylene monooxygenase, which catalyzes the multistep oxygenation of one methyl group of toluene and xylenes to corresponding alcohols, aldehydes, and acids. A fed-batch based two-liquid phase bioconversion was established with bis(2-ethylhexyl)- phthalate as organic carrier solvent and a phase ratio of 0.5; the product formation pattern, the impact of the nutrient feeding strategy, and the partitioning behavior of the reactants were studied. On the basis of the favorable conditions provided by the two-liquid phase system, engineering of the initial pseudocumene concentration allowed exploiting the complex kinetics of the multistep reaction for the exclusive production of 3,4-dimethyl- benzaldehyde. Further oxidation of the product to 3,4-dimethylbenzoic acid could be inhibited by suitable concentrations of pseudocumene or 3,4-dimethylbenzyl alcohol. The optimized biotransformation setup includes a completely defined medium with high iron content and a nutrient feeding strategy that avoids severe glucose limitation as well as high inhibitory glucose levels. Using such a system on a 2-liter scale, we were able to produce, within 14.5 h, 30 g of 3,4-dimethylbenzaldehyde as predominant reactant in the organic phase and reached a maximal productivity of 1.6 g per liter liquid volume per hour. The present study implicates that the two-liquid phase concept is an efficient tool to exploit the kinetics of multistep biotransformations in general.     

Detection of alkanes, alcohols, and aldehydes using bioluminescence

We report a novel method for the rapid, sensitive, and quantitative detection of alkanes, alcohols, and aldehydes that relies on the reaction of bacterial luciferase with an aldehyde, resulting in the emission of light. Primary alcohols with corresponding aldehydes that are within the substrate range of the particular luciferase are detected after conversion to the aldehyde by an alcohol dehydrogenase. In addition, alkanes themselves may be detected by conversion to primary alcohols by an alkane hydroxylase, followed by conversion to the aldehyde by alcohol dehydrogenase. We developed a rapid bioluminescent method by genetically engineering the genes encoding bacterial luciferase, alcohol dehydrogenase, and alkane hydroxylase into a plasmid for simultaneous expression in an E. coli host cell line. Alkanes, alcohols, or aldehydes were detected within seconds, with sensitivity in the micromolar range, by measuring the resulting light emission with a microplate reader. We demonstrate the application of this method for the detection of alkanes, alcohols, and aldehydes and for the detection of alkane hydroxylase and alcohol dehydrogenase activity in vivo. This method is amenable to the high-throughput screening needs required for the identification of novel catalysts.     

Outer membrane protein AlkL boosts biocatalytic oxyfunctionalization of hydrophobic substrates in Escherichia coli

The outer membrane of microbial cells forms an effective barrier for hydrophobic compounds, potentially causing an uptake limitation for hydrophobic substrates. Low bioconversion activities (1.9 U g(cdw)(-1)) have been observed for the ω-oxyfunctionalization of dodecanoic acid methyl ester by recombinant Escherichia coli containing the alkane monooxygenase AlkBGT of Pseudomonas putida GPo1. Using fatty acid methyl ester oxygenation as the model reaction, this study investigated strategies to improve bacterial uptake of hydrophobic substrates. Admixture of surfactants and cosolvents to improve substrate solubilization did not result in increased oxygenation rates. Addition of EDTA increased the initial dodecanoic acid methyl ester oxygenation activity 2.8-fold. The use of recombinant Pseudomonas fluorescens CHA0 instead of E. coli resulted in a similar activity increase. However, substrate mass transfer into cells was still found to be limiting. Remarkably, the coexpression of the alkL gene of P. putida GPo1 encoding an outer membrane protein with so-far-unknown function increased the dodecanoic acid methyl ester oxygenation activity of recombinant E. coli 28-fold. In a two-liquid-phase bioreactor setup, a 62-fold increase to a maximal activity of 87 U g(cdw)(-1) was achieved, enabling the accumulation of high titers of terminally oxyfunctionalized products. Coexpression of alkL also increased oxygenation activities toward the natural AlkBGT substrates octane and nonane, showing for the first time clear evidence for a prominent role of AlkL in alkane degradation. This study demonstrates that AlkL is an efficient tool to boost productivities of whole-cell biotransformations involving hydrophobic aliphatic substrates and thus has potential for broad applicability.     

Suitability of recombinant Escherichia coli and Pseudomonas putida strains for selective biotransformation of m-nitrotoluene by xylene monooxygenase

Escherichia coli JM101(pSPZ3), containing xylene monooxygenase (XMO) from Pseudomonas putida mt-2, catalyzes specific oxidations and reductions of m-nitrotoluene and derivatives thereof. In addition to reactions catalyzed by XMO, we focused on biotransformations by native enzymes of the E. coli host and their effect on overall biocatalyst performance. While m-nitrotoluene was consecutively oxygenated to m-nitrobenzyl alcohol, m-nitrobenzaldehyde, and m-nitrobenzoic acid by XMO, the oxidation was counteracted by an alcohol dehydrogenase(s) from the E. coli host, which reduced m-nitrobenzaldehyde to m-nitrobenzyl alcohol. Furthermore, the enzymatic background of the host reduced the nitro groups of the reactants resulting in the formation of aromatic amines, which were shown to effectively inhibit XMO in a reversible fashion. Host-intrinsic oxidoreductases and their reaction products had a major effect on the activity of XMO during biocatalysis of m-nitrotoluene. P. putida DOT-T1E and P. putida PpS81 were compared to E. coli JM101 as alternative hosts for XMO. These promising strains contained an additional dehydrogenase that oxidized m-nitrobenzaldehyde to the corresponding acid but catalyzed the formation of XMO-inhibiting aromatic amines at a significantly lower level than E. coli JM101.     

Characterization of cinnamyl alcohol dehydrogenase of Helicobacter pylori. An aldehyde dismutating enzyme

Cinnamyl alcohol dehydrogenases (CAD; 1.1.1.195) catalyse the reversible conversion of p-hydroxycinnamaldehydes to their corresponding alcohols, leading to the biosynthesis of lignin in plants. Outside of plants their role is less defined. The gene for cinnamyl alcohol dehydrogenase from Helicobacter pylori (HpCAD) was cloned in Escherichia coli and the recombinant enzyme characterized for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterized plant enzymes. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not been shown previously for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell.     

Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells

Whole cells expressing the non-heme diiron hydroxylases AlkB and toluene 4-monooxygenase (T4MO) were used to probe enzyme reaction mechanisms. AlkB catalyzes the hydroxylation of the radical clock substrates bicyclo[4.1.0]heptane (norcarane), spirooctane and 1,1-diethylcyclopropane, and does not catalyze the hydroxylation of the radical clocks 1,1-dimethylcyclopropane or 1,1,2,2-tetramethylcyclopropane. The hydroxylation of norcarane yields a distribution of products consistent with an "oxygen-rebound" mechanism for the enzyme in both the wild type Pseudomonas putida GPo1 and AlkB from P. putida GPo1 expressed in Escherichia coli. Evidence for the presence of a substrate-based radical during the reaction mechanism is clear. With norcarane, the lifetime of that radical varies with experimental conditions. Experiments with higher substrate concentrations yield a shorter radical lifetime (approximately 1 ns), while experiments with lower substrate concentrations yield a longer radical lifetime (approximately 19 ns). Consistent results were obtained using either wild type or AlkB-equipped host organisms using either "resting cell" or "growing cell" approaches. T4MO expressed in E. coli also catalyzes the hydroxylation of norcarane with a radical lifetime of approximately 0.07 ns. No radical lifetime dependence on substrate concentration was seen. Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropane, and diethylcyclopropane are consistent with a restricted active site for AlkB.     

Identification of novel hemes generated by heme A synthase: evidence for two successive monooxygenase reactions

Heme A, an obligatory cofactor in eukaryotic cytochrome c oxidase, is produced from heme B (protoheme) via two enzymatic reactions catalyzed by heme O synthase and heme A synthase. Heme O synthase is responsible for the addition of a farnesyl moiety, while heme A synthase catalyzes the oxidation of a methyl substituent to an aldehyde. We have cloned the heme O synthase and heme A synthase genes from Bacillus subtilis (ctaB and ctaA) and overexpressed them in Escherichia coli to probe the oxidative mechanism of heme A synthase. Because E. coli does not naturally produce or utilize heme A, this strategy effectively decoupled heme A biosynthesis from the native electron transfer pathway and heme A transport, allowing us to observe two previously unidentified hemes. We utilized HPLC, UV/visible spectroscopy, and tandem mass spectrometry to identify these novel hemes as derivatives of heme O containing an alcohol or a carboxylate moiety at position C8 on pyrrole ring D. We interpret these derivatives to be the putative alcohol intermediate and an overoxidized byproduct of heme A synthase. Because we have shown that all hemes produced by heme A synthase require O(2) for their synthesis, we propose that heme A synthase catalyzes the oxidation of the C8 methyl to an aldehyde group via two discrete monooxygenase reactions.     

Oxidation of both termini of p- and m-xylene by Escherichia coli transformed with xylene monooxygenase gene

Xylene monooxygenase (XMO) from Pseudomonas putida mt-2 catalyzes oxidation of methyl group of toluene and xylenes. While it has been postulated that this enzyme oxidizes one methyl group of xylene, we observed that both methyl groups in p- and m-xylene were oxidized to alcohol and aldehyde when the relevant genes (xylM and xylA) were co-expressed in Escherichia coli C600 and MC4100. When p-xylene was used as a substrate, p-hydroxymethylbenzaldehyde and p-xylyleneglycol were identified, in addition to p-methylbenzylalcohol and p-tolualdehyde. When m-xylene was used as a substrate, m-hydroxymethylbenzaldehyde and m-xylyleneglycol were identified, in addition to m-methylbenzylalcohol and m-tolualdehyde. Ratio of the products varied significantly according to the reaction condition and host strain, presumably reflecting the relative activity of XMO and host-derived dehydrogenase(s). Using various oxidized compounds as substrates, it was indicated that dialcohol (p- or m-xylyleneglycol) was formed via p- or m-hydroxymethylbenzaldehyde, respectively, rather than directly from corresponding monoalcohol (p- or m-methybenzylalcohol).     

Altered mechanism of the alkanesulfonate FMN reductase with the monooxygenase enzyme

The two-component alkanesulfonate monooxygenase system from Escherichia coli is comprised of an FMN reductase (SsuE) and a monooxygenase enzyme (SsuD) that together catalyze the oxidation of alkanesulfonate to the corresponding aldehyde and sulfite products. To determine the effects of protein interactions on catalysis, the steady-state kinetic parameters for SsuE were determined in single-enzyme assays and in the presence of the monooxygenase enzyme and alkanesulfonate substrate. In single-enzyme kinetic assays, SsuE followed an ordered sequential mechanism, with NADPH as the first substrate to bind and NADP+ as the last product to dissociate. However, in the presence of SsuD and octanesulfonate the kinetic mechanism of SsuE is altered to a rapid equilibrium ordered mechanism, and the Km value for FMN is increased 10-fold. These results suggest that both the SsuD enzyme and alkanesulfonate substrate are required to ensure that the FMN reductase reaction proceeds to form the ternary complex with the subsequent generation of reduced flavin transfer.     

The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation

Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.     

Characterization of a fatty acyl-CoA reductase from Marinobacter aquaeolei VT8: a bacterial enzyme catalyzing the reduction of fatty acyl-CoA to fatty alcohol

Fatty alcohols are of interest as a renewable feedstock to replace petroleum compounds used as fuels, in cosmetics, and in pharmaceuticals. One biological approach to the production of fatty alcohols involves the sequential action of two bacterial enzymes: (i) reduction of a fatty acyl-CoA to the corresponding fatty aldehyde catalyzed by a fatty acyl-CoA reductase, followed by (ii) reduction of the fatty aldehyde to the corresponding fatty alcohol catalyzed by a fatty aldehyde reductase. Here, we identify, purify, and characterize a novel bacterial enzyme from Marinobacter aquaeolei VT8 that catalyzes the reduction of fatty acyl-CoA by four electrons to the corresponding fatty alcohol, eliminating the need for a separate fatty aldehyde reductase. The enzyme is shown to reduce fatty acyl-CoAs ranging from C8:0 to C20:4 to the corresponding fatty alcohols, with the highest rate found for palmitoyl-CoA (C16:0). The dependence of the rate of reduction of palmitoyl-CoA on substrate concentration was cooperative, with an apparent K(m) ~ 4 μM, V(max) ~ 200 nmol NADP(+) min(-1) (mg protein)(-1), and n ~ 3. The enzyme also reduced a range of fatty aldehydes with decanal having the highest activity. The substrate cis-11-hexadecenal was reduced in a cooperative manner with an apparent K(m) of ~50 μM, V(max) of ~8 μmol NADP(+) min(-1) (mg protein)(-1), and n ~ 2.     

O2 incorporation into a long-chain fatty acid during bacterial luminescence

The bioluminescence-dependent oxidation of a long-chain fatty aldehyde catalyzed by luciferase from Photobacterium phosphoreum has been studied in ¹⁸O₂ experiments. The results show the incorporation of one atom of molecular oxygen into the product, the corresponding fatty acid. This incorporation is not the result of exchange of ¹⁸O₂ with the aldehyde prior to oxidation to the acid, thereby indicating that the bacterial luciferase catalyzes an aldehyde monooxygenase reaction which is coupled with bioluminescence.     

Enhanced stress tolerance in Escherichia coli and Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline dehydrogenase fusion protein

In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5-fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector-transformed control plants.     

Oxidation of monohalogenated ethanes and n-chlorinated alkanes by whole cells of Nitrosomonas europaea.

We have investigated the substrate specificity of ammonia monooxygenase in whole cells of the nitrifying bacterium Nitrosomonas europaea for a number of aliphatic halogenated hydrocarbons. To determine the effect of the halogen substituent and carbon chain length on substrate reactivity, we measured the rates of oxidation of the monohalogenated ethanes (fluoroethane, chloroethane, bromoethane, and iodoethane) and n-chlorinated C1 to C4 alkanes by whole cells of N. europaea. For monohalogenated ethanes, acetaldehyde was the major organic product and little or none of any of the alternate predicted products (2-halogenated alcohols) were detected. The maximum rate of haloethane oxidation increased with decreasing halogen molecular weight from iodoethane to chloroethane (19 to 221 nmol/min per mg of protein). In addition, the amount of substrate required for the highest rate of haloethane oxidation increased with decreasing halogen molecular weight. For the n-chlorinated alkanes, the rate of dechlorination, as measured by the appearance of the corresponding aldehyde product, was greatest for chloroethane and decreased dramatically for chloropropane and chlorobutane (118, 4, and 8 nmol of aldehyde formed per min per mg of protein, respectively). The concentration profiles for halocarbon oxidation by ammonia monooxygenase showed apparent substrate inhibition when ammonia was used as the reductant source. When hydrazine was used as the electron donor, no substrate inhibition was observed, suggesting that the inhibition resulted from reductant limitation.     

Ternary complexes of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent system for probing the role of binding interactions with NAD(+) and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes the transfer of hydride ion from an alcohol substrate to the NAD(+) cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The enzyme is also an excellent catalyst for the reverse reaction. X-ray crystallography has shown that the NAD(+) binds in an extended conformation with a distance of 15 A between the buried reacting carbon of the nicotinamide ring and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-averaged models. Significantly, entries in the protein data bank contain both coordinates as well as temperature factors. However, enzyme function involves both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the vibrations of certain atoms in the active site where reactions take place. Ternary complexes produced during the reaction of the enzyme binary entity, E-NAD(+), with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolized via the E-NAD(+)-retinal ternary complex to retinoic acid (vitamin A acid). To unravel the mechanistic aspects of these transformations, the kinetics and energetics of interconversion between various ternary complexes are characterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Secondary kinetic isotope effects with retinol are not particularly large with the wild-type form of alcohol dehydrogenase from horse liver. We analyze alcohol dehydrogenase catalysis through a re-examination of the reaction coordinates. The ground states of the binary and ternary complexes are shown to be related to the corresponding transition states through topology and free energy acting along the reaction path.     

Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.     

Drosophila alcohol dehydrogenase: acetate-enzyme interactions and novel insights into the effects of electrostatics on catalysis

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones and that is also able to further oxidize aldehydes to their corresponding carboxylic acids. The structure of the ternary enzyme-NADH-acetate complex of the slow alleloform of Drosophila melanogaster ADH (DmADH-S) was solved at 1.6 A resolution by X-ray crystallography. The coenzyme stereochemistry of the aldehyde dismutation reaction showed that the obtained enzyme-NADH-acetate complex reflects a productive ternary complex although no enzymatic reaction occurs. The stereochemistry of the acetate binding in the bifurcated substrate-binding site, along with previous stereochemical studies of aldehyde reduction and alcohol oxidation shows that the methyl group of the aldehyde in the reduction reaction binds to the R1 and in the oxidation reaction to the R2 sub-site. NMR studies along with previous kinetic studies show that the formed acetaldehyde intermediate in the oxidation of ethanol to acetate leaves the substrate site prior to the reduced coenzyme, and then binds to the newly formed enzyme-NAD+ complex. Here, we compare the three-dimensional structure of D.melanogaster ADH-S and a previous theoretically built model, evaluate the differences with the crystal structures of five Drosophila lebanonensis ADHs in numerous complexed forms that explain the substrate specificity as well as subtle kinetic differences between these two enzymes based on their crystal structures. We also re-examine the electrostatic influence of charged residues on the surface of the protein on the catalytic efficiency of the enzyme.     

Stereoselective hydrogen abstraction by galactose oxidase

The fungal enzyme galactose oxidase is a radical copper oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on galactose oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state for substrate oxidation. In this work, these detailed mechanistic studies have been extended using a series of stereospecifically monodeuterated substrates, including 1-O-methyl-alpha-D-galactose as well as unsubstituted benzyl alcohol and 3- and 4-methoxy and 4-nitrobenzyl derivatives. Synthesis of all of these substrates was based on oxidation of the alpha,alpha'-dideuterated alcohol to the corresponding (2)H-labeled aldehyde, followed by asymmetric hydroboration using alpha-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by mass spectrometry to quantitatively evaluate the substrate dependence of the stereoselectivity of the catalytic reaction. For all of these substrates, the selectivity for pro-S hydrogen abstraction was at least 95%. This selectivity appears to be a direct consequence of constraints imposed by the enzyme on the orientation of substrates bearing a branched beta-carbon. Steady state analysis of kinetic isotope effects on V/K has resolved individual contributions from primary and alpha-secondary kinetic isotope effects in the reaction, providing a test for the involvement of an electron transfer redox equilibrium in the oxidation process. Multiple isotope effect measurements utilizing simultaneous labeling of the substrate and solvent have contributed to refinement of the relation between proton transfer and hydrogen atom transfer steps in substrate oxidation.