I311Membrane Bound Alcohol Dehydrogenase Adh Iii

Quinohemoprotein alcohol dehydrogenase (ADH III) is localized on the outer surface of the cytoplasmic membrane of the acetic acid bacteria, Acetobacter and Gluconobacter. ADH III has the most important role in vinegar production in oxidizing ethanol to acetaldehyde. The acetaldehyde generated is immediately oxidized to acetate by aldehyde dehydrogenase located close to ADH III on the same cytoplasmic membrane. As mentioned elsewhere, most people still believe that ethanol oxidation during...

Human Metabolites

An extraordinarily broad substrate range and very diverse reactions catalyzed by eukaryotic P450s make them attractive for pharmaceutical research. However, the low stability and activity of these enzymes hinder their application for drug synthesis. Implementation of more stable and active bacterial P450s, which are able to accept substrates of the mammalian counterparts, would open up a route to the production of new pharmaceuticals. Wild-type P450BM-3 (fatty acid hydroxylase) does not...

Gluconate Dehydrogenase Membrane Bound

D-Gluconate dehydrogenase (GADH) (EC 1.1.99.3) occurs on the outer surface of cytoplasmic membranes of aerobic bacteria, such as Pseudomonas, Klebsiella, Ser-ratia, and acetic acid bacteria. The enzyme activity is linked to the electron transport chain in the cytoplasmic membrane constituting a D-gluconate oxidase system 89-91 . GADH of P. aeruginosa shows a single protein band on native PAGE, but the enzyme preparations from K. Klebsiella and S. marcescens are separated into two protein bands...

New Mediators

As discussed in Section 2.2.1.7, improved mediators are needed to further develop viable laccase catalytic systems. To do this, extensive work has been carried out to test derivatives of promising known mediators, such as ABTS, HBT, and phe-nothiazine-10-propionic acid, to systematically study their structure-function relationship 30, 31, 136, 144, 175 . Phenols with extended conjugation and or steric structure causing low pKa have been studied to better balance their reactivity towards laccase...

Unexplored Type I BVMOs

In recent years, several type I BVMO genes have been identified that are associated with various specific microbial anabolic or catabolic pathways. Few biochemical data exist on these enzymes. However, based on the elucidated biosynthesis or degradation pathways, the identity of the physiological substrate of these type I BVMOs can be deduced as will be discussed below. Fumonisin toxins are a potentially serious problem as they are frequently found as contaminants in maize and maize-based food...

EDTA Treatment of the Membrane Fraction Carrying PQQ as Coenzyme

After a membrane suspension (10 mg of protein per mL) is mixed with 20 mmol L-1 EDTA for 30 min in an ice bath. The excess EDTA is removed from the membrane by ultracentrifugation two or three times at 68 000 x g for 60 min. The precipitate is resuspended in a buffer to wash EDTA out of the membrane fraction, followed by ultracentrifugation again under the same conditions. The resulting precipitate is resuspended with the same buffer. Under these conditions, as mentioned below, many...

P450cam and its Reductase System

The most intensively studied class I P450 redox system is P450cam along with its PdR and Pd redox partners. All components of the system are soluble proteins and interact with one another in the cytosol of Pseudomonas putida. The atomic structures of all three components of the system have been determined, and the P450 component has been structurally characterized in a number of different substrate- and ligand-bound forms e.g. 9, 10, 20, 21 . PdR is a 48 kDa FAD-binding protein which is reduced...

Quinate Oxidation Membrane Bound Quinate Dehydrogenase QDH

Unlike enzymes relating to the shikimate pathway found in the cytoplasm of microorganisms, the first report of a quinate-oxidizing enzyme in acetic acid bacteria was done by Whiting and Coggins 76 . They described quinate oxidation to 3-dehydroquinate (DQA) by an NAD (P)-independent quinate dehydrogenase (QDH) (EC 1.1.99.25) and shikimate (SKA) oxidation to 3-dehydroshikimate (DSA) by an enzyme associated with the particulate enzyme in the cytoplasmic membrane. van Kleef and Duine described the...

Soluble Cytochromes P450

The unquestionable advantages of the use of recombinant bacteria such as Escherichia coli are of course rapid cell growth and (at least in principle) high expression levels, putting these organisms at the forefront of genetically modified organisms used in industry. Thus, bacteria are the obvious choice as expression systems for soluble cytochromes P450, even more so as these are generally of bacterial origin. Arguably, the most widely studied soluble P450 for biotechnolo-gical application...

The Scope of P450 Engineering

It is not surprising that the engineering of bacterial P450 systems has been focused on the best characterized systems, P450cam and P450BM-3. There have been significant successes from structure-based redesign, site saturation mutagenesis, and directed evolution by error-prone PCR as well as chimera construction 90-92 . All these methods have limitations but the results to date have been very promising. Furthermore, these approaches are increasingly being combined to further enhance activity...

Laccase Based Defense Against Biological and Chemical Warfare Agents

The 2001 anthrax letter cases in the USA, and the 1995 sarin attack in Tokyo illustrate the threat that biological and chemical weapons pose when used by terrorists. They also emphasize how important it is for security, emergency, and medical agencies to be adequately equipped to decontaminate large, open areas such as subways, airports, and other public facilities when attacked by biological or chemical warfare agents. Traditionally used decontamination systems are not suited for such tasks,...

Mechanistic and Structural Properties of Type I BVMOs

The best-studied BVMO is cyclohexanone monooxygenase (CHMO) from Acineto-bacter sp. NCIMB 9871. This type I BVMO was first isolated in 1976 29 which set off many biocatalytic studies involving this monooxygenase 28 . Over the years a large number of different substrates (> 100) have been reported for this bacterial monooxygenase. The enzyme has been used as prototype BVMO to show that these enzymes are not only capable of performing Baeyer-Villiger oxidations but can also oxygenate sulfides,...

P450BM3 and Related CPR Fusion Enzymes

Armand Fulco's studies of fatty acid hydroxylase activity in the soil bacterium Bacillus megaterium revealed the first major P450 redox system deviating from the aforementioned class I and class II types 14 . The hydroxylase activity was found to be associated with a phenobarbital-inducible 119 kDa protein, which was then shown to be a P450 N-terminal fused to a CPR module C-terminal 68, 69 . The enzyme P450BM-3 or CYP102A1 is a soluble enzyme and a membrane anchor is absent from both the P450...

Other Routes to Driving P450 Catalytic Function

The fact that most P450 enzymes require at least one redox partner protein is often seen as a complicating factor in their exploitation for biotechnological applications. However, there are means to drive P450 catalysis that do not rely on exogenous protein partners. The best known of these is the peroxide shunt mechanism as illustrated in Fig. 5.2 whereby the resting state of the P450 can be converted to the ferric hydroperoxy form by reaction with hydrogen peroxide or organic peroxides . This...

Natural and Artificial P450Redox Partner Fusion Enzymes and their Biocatalytic Potential

The biotechnological advantages offered by the fusion of P450s to redox partner enzymes appear clear from studies on the P450BM-3 system. BM-3 has an efficient electron transport chain that enables fatty acid hydroxylation at rates 100-fold faster than many eukaryotic P450s. In addition, it is catalytically self-sufficient, obviating the requirement for isolation of 2-3 different enzymes and determining ratios of redox partner-to-P450 that give optimal turnover 14, 72 . The same advantages are...

Novel Laccase Catalytic Systems 2311 New Laccases

Besides the archetypical laccases discussed in Section 2.1.1, various seemingly unconventional laccases or analogs have been reported. Some of the isolated laccases with sequences highly homologous to typical multi-Cu oxidases appear as yellow or white when purified, indicating the lack of a canonical blue T1 Cu site. The site might be modified by oxidized substrates, as proposed for the Pleurotus ostreatus or Panus tigrinus yellow laccase 157, 158 , or contain other metal ions for example, Zn2...

Terpenoid Compounds

Terpenoid compounds constitute one of the largest groups of biological molecules. The parent terpene hydrocarbons are often readily available and their oxygenation gives derivatives which are fragrance and flavoring compounds, insect pheromones, and precursors to pharmaceuticals. The monoterpene -a-pinene is structurally related to camphor. The Y96F mutant of P450cam showed strengthened monoterpene binding and increased oxidation rates and coupling 82 . Pinene was oxidized mainly to verbenol...

Novel Synthetic Applications

Using various mediators, laccase may drive new, green synthetic applications in addition to those discussed in Section 2.2.1.5 2 . Laccase-oxidized N O mediators may abstract H from allylic C H to form allylic O, which may result in the homolysis of adjacent C O, C H, or C C bonds, or in the rearrangement of C C bonds. Under laccase oxidation, benzylic or aliphatic allylic hydrocarbons may be converted to benzylic or allylic alcohol, alkene, or cyclized products 2, 145, 156, 208, 222 . Toluene,...

Alkanes and Alicyclics

Engineering P450 enzymes into an alkane hydroxylase is an area of ongoing interest. A methane-oxidizing P450 enzyme may have important applications in the energy and chemicals sector while terminal oxidation of medium and long-chain alkanes and alkenes leads to fine chemicals. The CYP102 family of medium-chain fatty acid hydroxylases is a natural choice for engineering for alkane oxidation. Wild-type P450BM-3 showed low activity for octane oxidation 80 min-1 . The A74G F87V L188Q mutant...

General Features of P450cam and P450BM3

P450cam catalyzes the stereospecific oxidation of camphor to 5-exo-hydroxycam-phor Scheme 4.1 . It was the first P450 enzyme to be structurally characterized 26, 27 , identifying the amino acid side-chains that define the active site and contact the substrate. Camphor is bound by numerous non-covalent contacts and a hydrogen bond between the camphor carbonyl and the phenol side-chain of Y96, leading to a specific orientation with C5 directly above the heme iron Fig. 4.1 . Atkins and Sligar were...

Aldehyde Dehydrogenase Membrane Bound

Membrane-bound aldehyde dehydrogenase ALDH in acetic acid bacteria acts on a wide range of aliphatic aldehydes except for formaldehyde. Aldehydes with a carbon chain length of 2-4 are oxidized most rapidly with ALDH from both genera of Acetobacter and Gluconobacter. The enzyme is localized on the outer surface of the cytoplasmic membrane of the organisms and has a close topological and functional relation to ADH III. Aldehyde oxidation is linked to the respiratory chain as described for the...

Membrane Bound DGlucose Dehydrogenase mGDH

The quinoprotein D-glucose dehydrogenase EC 1.1.99.17 GDH occurs on the outer surface of the cytoplasmic membrane of oxidative bacteria such as Pseudomonas and Gluconobacter strains and catalyzes direct oxidation of D-glucose to D-gluconate via D-glucono-S-lactone membrane-bound D-glucose dehydrogenase, m-GDH . It is known as an alternative pathway to the phosphotransferase system of bacteria to catalyze D-glucose assimilation. m-GDH is found in a variety of bacteria including Gram-negative...

Fructose Dehydrogenase Membrane Bound

D-Fructose oxidation to 5-keto-D-fructose 5KF is catalyzed by membrane-bound fructose dehydrogenase FDH, EC 1.1.99.11 , which contains a covalently bound FAD as the primary coenzyme. FDH was purified from the membrane fraction of G. industrius IFO 3260. FDH was solubilized from the membrane with 1 Triton X-100 and further purified to a homogeneous state 86 . The purified FDH had a sedimentation coefficient of 5.8S and the total molecular mass was estimated to be 140 kDa by gel filtration. The...

Arabitol Dehydrogenase Membrane Bound

Because there is little information about C-5 sugar alcohol oxidation, evidence has been presented confirming that L-ribulose formation, the oxidation product of ribitol, was catalyzed by the action of a membrane-bound PQQ-dependent ribitol dehydrogenase, but not by a cytosolic NAD-dependent ribitol dehydrogenase 50 . Due to the high hydrophobicity and instability of the enzyme for pen-titol oxidation, its solubilization from the membrane and purification remained to be achieved. Several...

Cyclic Alcohol Dehydrogenase Secondary Alcohol Dehydrogenase Membrane Bound

Aliphatic secondary alcohol Aliphatic ketone Numerous investigations on microbial and enzymatic oxidation of cyclic alcohols have been carried out with various bacteria. Most of them are related to NAD-dependent cyclohexanol dehydrogenase EC 1.1.1.245 from pseudomonads and Acinetobacter sp. 23-26 . Microbial cyclopentanol oxidation was also studied in Pseudomonas sp. and NAD-dependent cyclopentanol dehydrogenase EC 1.1.1.163 was indicated as the responsible enzyme in the first step of the...