Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

Amperometric determination of lactate with novel trienzyme/poly(carbamoyl) sulfonate hydrogel-based sensor

A novel trienzyme sensor for the amperometric determination of lactate was constructed by immobilizing salicylate hydroxylase (SHL, E.C. 1.14.13.1), l-lactate dehydrogenase (LDH, E.C. 1.1.1.27), and pyruvate oxidase (PyOD, E.C. 1.2.3.3) on a Clark-type oxygen electrode. The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. LDH catalyzes the specific dehydrogenation of lactate consuming NAD(+). SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate in the presence of oxygen and NADH produced by LDH. PyOD decarboxylates pyruvate using oxygen and phosphate. SHL and PyOD force the equilibrium of dehydrogenation of lactate by LDH to the product side by consuming NADH and pyruvate, respectively. Dissolved oxygen acts as an essential material for both PyOD and SHL during their respective enzymatic reactions. Therefore, an amplified signal, caused by the consumptions of dissolved oxygen by the two enzymes, was observed in the measurement of lactate. Regeneration of cofactor was found in the trienzyme system. A Teflon membrane was used to fabricate the sensor in order to avoid interferences. The sensor has a fast response (2s) and short recovery times (2 min). The total test time for a measurement by using this lactate sensor (4 min) was faster than using a commercial lactate testing kit (up to 10 min). The sensor has a linear range between 10 and 400 microM lactate, with a detection limit of 4.3 microM. A good agreement (R2 = 0.9984) with a commercial lactate testing kit was obtained in beverage sample measurements.     

Synthesis and characterization of homochiral polymeric S-malato molybdate(VI): toward the potentially stereospecific formation and absolute configuration of iron-molybdenum cofactor in nitrogenase

Reaction of sodium or potassium molybdate and excess malic acid in a wide range of pH values (pH 4.0–7.0) resulted in the isolation of two cis-dioxo-bis(malato)-Mo(VI) complexes, viz. Na₃[MoO₂H(S-mal)₂] and K₃[MoO₂H(S-mal)₂]·H₂O (H₃mal=malic acid). The sodium complex is also characterized by an X-ray structure analysis, showing that the mononuclear Mo units are linked together via very strong symmetric CO₂···H··· O₂C-hydrogen bond [2.432(5) Å], forming a polymeric chain. The molybdenum atoms are quasi-octahedrally coordinated by two cis-oxo groups and two bidentate malate ligands via its alkoxy and -carboxyl groups, while the β-carboxylic and carboxylate groups remain uncomplexed, as the coordination of vicinal carboxylate and alkoxide of homocitrate in FeMo cofactor of nitrogenase. The absolute configuration of the metal center in this S-malato complex is assigned as Λ and the homochirality within the chain is established as a homochiral form ···Λ_S–Λ_S–Λ_S–Λ_S···. It is proposed that the chiral configuration of the metal center in wild-type FeMo-co biosynthesis might be induced by the early coordination of the chiral R-homocitric acid, while a mixture of raceme might be obtained in the biosynthesis of NifV⁻ FeMo-cofactor. The absolute configuration of wild-type FeMo-cofactor is assigned as Δ_R.     

Structural and functional analysis of the riboflavin synthesis genes encoding GTP cyclohydrolase II (ribA), DHBP synthase (ribBA), riboflavin synthase (ribC), and riboflavin deaminase/reductase (ribD) from Helicobacter pylori strain P1

The functions of the riboflavin synthesis gene homologues ribA, ribBA, ribC, and ribD from Helicobacter pylori strain P1 were confirmed by complementation of defined Escherichia coli mutant strains. The H. pylori ribBA gene, which is similar to bifunctional ribBA genes of Gram-positive bacteria, fully complemented the ribB mutation and partially restored growth in a ribC mutant. However, ribBA did not complement the ribA mutation in E. coli, thus explaining the presence of the additional separate copy of the ribA gene in the H. pylori chromosome. In E. coli exclusively ribA conferred hemolytic activity and gave rise to production of molecules with fluorescence characteristics similar to flavins, as observed earlier. The E. coli hemolysin ClyA was not involved in causing the hemolytic phenotype. No riboflavin synthesis genes on plasmids conferred iron uptake functions to a siderophore-deficient mutant of E. coli. Marker exchange mutagenesis of the genes in H. pylori was not successful indicating that riboflavin synthesis is essential for basic metabolic functions of the gastric pathogen.     

Dual lifetime referencing enables pH‐control for oxidoreductions in hydrogel‐stabilized biphasic reaction systems

pH‐shifts are a serious challenge in cofactor dependent biocatalytic oxidoreductions. Therefore, a pH control strategy was developed for reaction systems, where the pH value is not directly measurable. Such a reaction system is the biphasic aqueous‐organic reaction system, where the oxidoreduction of hydrophobic substrates in organic solvents is catalysed by hydrogel‐immobilized enzymes, and enzyme‐coupled cofactor regeneration is accomplished via formate dehydrogenase, leading to a pH‐shift. Dual lifetime referencing (DLR), a fluorescence spectroscopic method, was applied for online‐monitoring of the pH‐value within the immobilizates during the reaction, allowing for a controlled dosage of formic acid. It could be shown that by applying trisodium 8‐hydroxypyrene‐1, 3, 6‐trisulfonate as pH indicator and Ru(II) tris(4, 7‐diphenyl‐1, 10‐phenantroline) (Ru[dpp]) as a reference luminophore the control of the pH‐value in a macroscopic gel‐bead‐stabilized aqueous/organic two phase system in a range of pH 6.5 to 8.0 is possible. An experimental proof of concept could maintain a stable pH of 7.5 ± 0.15 during the reaction for at least 105 h. With these results, it could be shown that DLR is a powerful tool for pH‐control within reaction systems with no direct access for conventional pH‐measurement.     

Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD⁺-dependent with a thermal optimum for activity at 55–60°C and an apparent midpoint melting temperature (T _m) of 70°C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD⁺-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD⁺-IDHs with high sequence identity (48–57%) to McIDH were however identified in a variety of bacteria showing that NAD⁺-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD⁺-IDH and the subfamily III of IDH was redefined to include bacterial NAD⁺- and NADP⁺-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD⁺-IDH are derived from the McIDH-like IDHs.     

Cofactor regeneration for sustainable enzymatic biosynthesis

Oxidoreductases are attractive catalysts for biosynthesis of chiral compounds and polymers, construction of biosensors, and degradation of environmental pollutants. Their practical applications, however, can be quite challenging since they often require cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These cofactors are generally expensive. Efficient regeneration of cofactors is therefore critical to the economic viability of industrial-scale biotransformations using oxidoreductases. The chemistry of cofactor regeneration is well known nowadays. The challenge is mostly regarding how to achieve the regeneration with immobilized enzyme systems which are preferred for industrial processes to facilitate the recovery and continuous use of the catalysts. This has become a great hurdle for the industrialization of many promising enzymatic processes, and as a result, most of the biotransformations involving cofactors have been traditionally performed with living cells in industry. Accompanying the rapidly growing interest in industrial biotechnology, immobilized enzyme biocatalyst systems with cofactor regeneration have been the focus for many studies reported since the late 1990s. The current paper reviews the methods of cofactor retention for development of sustainable and regenerative biocatalysts as revealed in these recent studies, with the intent to complement other reviewing articles that are mostly regeneration chemistry-oriented. We classify in this paper the methods of sustainable cofactor regeneration into two categories, namely membrane entrapment and solid-attachment of cofactors.     

The role of cysteine 160 in thiamine diphosphate binding of the Calvin-Benson-Bassham cycle transketolase of Rhodobacter sphaeroides

The transketolase gene (cbbT) that encodes the Calvin-Benson-Bassham pathway transketolase (CbbT) of Rhodobacter sphaeroides was overexpressed in Escherichia coli and the recombinant protein purified to homogeneity. Like other transketolases, R. sphaeroides CbbT was found to be inactivated in the presence of oxygen. At its optimal pH of 7.8, CbbT displays a specific activity of 37 U/mg, a KR5P of 949 microM, a KXu5P of 11 microM, and a KThDP of 1.8 microM. Cysteine 160, equivalent to Cys159 of the yeast enzyme, is found within the active site and is loosely conserved amongst several sources of transketolase. To investigate the role of cysteine 160 found in the active site of R. sphaeroides CbbT, this residue was targeted for mutagenesis. Cys160 was changed to alanine, serine, aspartate, and glutamate. To compare the effect of these mutations on ThDP binding, spectral techniques were employed in addition to analysis by enzymatic activity. Fluorescence quenching was used to measure both equilibrium binding constants as well as first order rates of binding. The results of these studies indicated that Cys160 played an important and substantial role in cofactor binding, revealing the importance of this loosely conserved residue. In addition, the Cys160 mutants did not appear to alter oxygen-mediated inactivation.     

An approach to the residence time distribution for stochastic multi-compartment models

Stochastic compartmental models are widely used in modeling processes such as drug kinetics in biological systems. This paper considers the distribution of the residence times for stochastic multi-compartment models, especially systems with non-exponential lifetime distributions. The paper first derives the moment generating function of the bivariate residence time distribution for the two-compartment model with general lifetimes and approximates the density of the residence time using the saddlepoint approximation. Then, it extends the distributional approach to the residence time for multi-compartment semi-Markov models combining the cofactor rule for a single destination and the analytic approach to the two-compartment model. This approach provides a complete specification of the residence time distribution based on the moment generating function and thus facilitates an easier calculation of high-order moments than the approach using the coefficient matrix. Applications to drug kinetics demonstrate the simplicity and usefulness of this approach.