Kirsten R. Wolthers

Microfluidics Integrated Biosensors: A Leading Technology towards Lab-on-a-Chip and Sensing Applications

A biosensor can be defined as a compact analytical device or unit incorporating a biological or biologically derived sensitive recognition element immobilized on a physicochemical transducer to measure one or more analytes. Microfluidic systems, on the other hand, provide throughput processing, enhance transport for controlling the flow conditions, increase the mixing rate of different reagents, reduce sample and reagents volume (down to nanoliter), increase sensitivity of detection, and utilize the same platform for both sample preparation and detection. In view of these advantages, the integration of microfluidic and biosensor technologies provides the ability to merge chemical and biological components into a single platform and offers new opportunities for future biosensing applications including portability, disposability, real-time detection, unprecedented accuracies, and simultaneous analysis of different analytes in a single device. This review aims at representing advances and achievements in the field of microfluidic-based biosensing. The review also presents examples extracted from the literature to demonstrate the advantages of merging microfluidic and biosensing technologies and illustrate the versatility that such integration promises in the future biosensing for emerging areas of biological engineering, biomedical studies, point-of-care diagnostics, environmental monitoring, and precision agriculture.

Large-scale Domain Dynamics and Adenosylcobalamin Reorientation Orchestrate Radical Catalysis in Ornithine 4,5-Aminomutase

d-Ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii converts d-ornithine to 2,4-diaminopentanoic acid by way of radical propagation from an adenosylcobalamin (AdoCbl) to a pyridoxal 5′-phosphate (PLP) cofactor. We have solved OAM crystal structures in different catalytic states that together demonstrate unusual stability of the AdoCbl Co-C bond and that radical catalysis is coupled to large-scale domain motion. The 2.0-Å substrate-free enzyme crystal structure reveals the Rossmann domain, harboring the intact AdoCbl cofactor, is tilted toward the edge of the PLP binding triose-phosphate isomerase barrel domain. The PLP forms an internal aldimine link to the Rossmann domain through Lys629, effectively locking the enzyme in this “open” pre-catalytic conformation. The distance between PLP and 5′-deoxyadenosyl group is 23 Å, and large-scale domain movement is thus required prior to radical catalysis. The OAM crystals contain two Rossmann domains within the asymmetric unit that are unconstrained by the crystal lattice. Surprisingly, the binding of various ligands to OAM crystals (in an oxygen-free environment) leads to transimination in the absence of significant reorientation of the Rossmann domains. In contrast, when performed under aerobic conditions, this leads to extreme disorder in the latter domains correlated with the loss of the 5′-deoxyadenosyl group. Our data indicate turnover and hence formation of the “closed” conformation is occurring within OAM crystals, but that the equilibrium is poised toward the open conformation. We propose that substrate binding induces large-scale domain motion concomitant with a reconfiguration of the 5′-deoxyadenosyl group, triggering radical catalysis in OAM.

Cobalamin uptake and reactivation occurs through specific protein interactions in the methionine synthase–methionine synthase reductase complex

Human methionine synthase reductase (MSR), a diflavin enzyme, restores the activity of human methionine synthase through reductive methylation of methionine synthase (MS)‐bound cob(II)alamin. Recently, it was also reported that MSR enhances uptake of cobalamin by apo‐MS, a role associated with the MSR‐catalysed reduction of exogenous aquacob(III)alamin to cob(II)alamin [Yamada K, Gravel RA, TorayaT & Matthews RG (2006) Proc Natl Acad Sci USA103, 9476–9481]. Here, we report the expression and purification of human methionine synthase from Pichia pastoris. This has enabled us to assess the ability of human MSR and two other structurally related diflavin reductase enzymes (cytochrome P450 reductase and the reductase domain of neuronal nitric oxide synthase) to: (a) stimulate formation of holo‐MS from aquacob(III)alamin and the apo‐form of MS; and (b) reactivate the inert cob(II)alamin form of MS that accumulates during enzyme catalysis. Of the three diflavin reductases studied, cytochrome P450 reductase had the highest turnover rate (55.5 s−1) for aquacob(III)alamin reduction, and the reductase domain of neuronal nitric oxide synthase elicited the highest specificity (kcat/Km of 1.5 × 105 m−1·s−1) and MSR had the lowest Km (6.6 μm) for the cofactor. Despite the ability of all three enzymes to reduce aquacob(III)alamin, only MSR (the full‐length form or the isolated FMN domain) enhanced the uptake of cobalamin by apo‐MS. MSR was also the only diflavin reductase to reactivate the inert cob(II)alamin form of purified human MS (Kact of 107 nm) isolated from Pichia pastoris. Our work shows that reactivation of cob(II)alamin MS and incorporation of cobalamin into apo‐MS is enhanced through specific protein–protein interactions between the MSR FMN domain and MS.

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

We report an analysis of the reaction mechanism of ornithine 4,5-aminomutase, an adenosylcobalamin (AdoCbl)- and pyridoxal l-phosphate (PLP)-dependent enzyme that catalyzes the 1,2-rearrangement of the terminal amino group of d-ornithine to generate (2R,4S)-2,4-diaminopentanoic acid. We show by stopped-flow absorbance studies that binding of the substrate d-ornithine or the substrate analogue d-2,4-diaminobutryic acid (DAB) induces rapid homolysis of the AdoCbl Co–C bond (781 s–1, d-ornithine; 513 s–1, DAB). However, only DAB results in the stable formation of a cob(II)alamin species. EPR spectra of DAB and [2,4,4-2H3]DAB bound to holo-ornithine 4,5-aminomutase suggests strong electronic coupling between cob(II)alamin and a radical form of the substrate analog. Loading of substrate/analogue onto PLP (i.e. formation of an external aldimine) is also rapid (532 s–1, d-ornithine; 488 s–1, DAB). In AdoCbl-depleted enzyme, formation of the external aldimine occurs over long time scales (∼50 s) and occurs in three resolvable kinetic phases, identifying four distinct spectral intermediates (termed A–D). We infer that these represent the internal aldimine (λmax 416 nm; A), two different unliganded PLP states of the enzyme (λmax at 409 nm; B and C), and the external aldimine (λmax 426 nm; D). An imine linkage with d-ornithine and DAB generates both tautomeric forms of the external aldimine, but with d-ornithine the equilibrium is shifted toward the ketoimine state. The influence of this equilibrium distribution of prototropic isomers in driving homolysis and stabilizing radical intermediate states is discussed. Our work provides the first detailed analysis of radical-based catalysis in this Class III AdoCbl-dependent enzyme.

Mitochondrial transcription factor A (Tfam) is a pro-inflammatory extracellular signaling molecule recognized by brain microglia.

Microglia represent mononuclear phagocytes in the brain and perform immune surveillance, recognizing a number of signaling molecules released from surrounding cells in both healthy and pathological situations. The microglia interact with several damage-associated molecular pattern molecules (DAMPs) and recent data indicate that mitochondrial transcription factor A (Tfam) could act as a specific DAMP in peripheral tissues. This study tested the hypothesis that extracellular Tfam induces pro-inflammatory and cytotoxic responses of the microglia. Three different types of human mononuclear phagocytes were used to model human microglia: human peripheral blood monocytes from healthy donors, human THP-1 monocytic cells, and human primary microglia obtained from autopsy samples. When combined with interferon (IFN)-γ, recombinant human Tfam (rhTfam) induced secretions that were toxic to human SH-SY5Y neuroblastoma cells in all three models. Similar cytotoxic responses were observed when THP-1 cells and human microglia were exposed to human mitochondrial proteins in the presence of IFN-γ. rhTfam alone induced expression of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and IL-8 by THP-1 cells. This induction was further enhanced in the presence of IFN-γ. Upregulated secretion of IL-6 in response to rhTfam plus IFN-γ was confirmed in primary human microglia. Use of specific inhibitors showed that the rhTfam-induced cytotoxicity of human THP-1 cells depended partially on activation of c-Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (MAPK). Overall, our data support the hypothesis that, in the human brain, Tfam could act as an intercellular signaling molecule that is recognized by the microglia to cause pro-inflammatory and cytotoxic responses.

Mutagenesis of a conserved glutamate reveals the contribution of electrostatic energy to adenosylcobalamin co-C bond homolysis in ornithine 4,5-aminomutase and methylmalonyl-CoA mutase.

Binding of substrate to ornithine 4,5-aminomutase (OAM) and methylmalonyl-CoA mutase (MCM) leads to the formation of an electrostatic interaction between a conserved glutamate side chain and the adenosyl ribose of the adenosylcobalamin (AdoCbl) cofactor. The contribution of this residue (Glu338 in OAM from Clostridium sticklandii and Glu392 in human MCM) to AdoCbl Co-C bond labilization and catalysis was evaluated by substituting the residue with a glutamine, aspartate, or alanine. The OAM variants, E338Q, E338D, and E338A, showed 90-, 380-, and 670-fold reductions in catalytic turnover and 20-, 60-, and 220-fold reductions in k(cat)/K(m), respectively. Likewise, the MCM variants, E392Q, E392D, and E392A, showed 16-, 330-, and 12-fold reductions in k(cat), respectively. Binding of substrate to OAM is unaffected by the single-amino acid mutation as stopped-flow absorbance spectroscopy showed that the rates of external aldimine formation in the OAM variants were similar to that of the native enzyme. The decrease in the level of catalysis is instead linked to impaired Co-C bond rupture, as UV-visible spectroscopy did not show detectable AdoCbl homolysis upon binding of the physiological substrate, d-ornithine. AdoCbl homolysis was also not detected in the MCM mutants, as it was for the native enzyme. We conclude from these results that a gradual weakening of the electrostatic energy between the protein and the ribose leads to a progressive increase in the activation energy barrier for Co-C bond homolysis, thereby pointing to a key role for the conserved polar glutamate residue in controlling the initial generation of radical species.

ELDOR spectroscopy reveals that energy landscapes in human methionine synthase reductase are extensively remodelled following ligand and partner protein binding.

modules, which bind, from the N to C terminus, respectively, homocysteine, methyltetrahydrofolate, cobalamin, and S-adenosylmethionine (SAM). The C-terminal “activation domain” (AD) also interacts with methionine synthase reductase (MSR), which is an NADPH-dependent diflavin oxidoreductase containing 1 mol equivalent of FAD and FMN. Occasionally, MS becomes inactivated through oxidation of cob(I)alamin to cob(II)alamin and reactivation by electron transfer from MSR is required. Direct reduction of MS by NADPH via FAD and FMN is thermodynamically not feasible because the redox potential of cob(I/II)alamin is far more negative than that of NADP . Consequently, Nature traps the small amounts of cob(I)alamin in equilibrium with cob(II)alamin by irreversible methylation with SAM (Scheme 1). The inferred multidomain structure of MSR and the presence of a large linker between the component flavin-containing domains suggests a large degree of conformational flexibility in the protein. By analogy with other diflavin oxidoreductases related to MSR, such as nitric oxide synthase and cytochrome P450 reductase, it has been suggested that MSR fluctuates between “closed” and “open” conformations. In this simple model, one conformation (“open” conformation) of MSR presents the FMN domain so that it is available for interaction with the AD of MS; in another conformation (a “closed” conformation) the FMN domain is in close proximity to the NADPH/FAD-binding domain, where it facilitates interflavin electron transfer (Figure 1). Although this model has not been tested explicitly, these multiple conformations would optimise electronic coupling to the FAD domain (closed conformation) and cob(II)alamin (open conformation) and require a “swinging” motion for the FMN domain between the two states. Such motion would, therefore, define an energy landscape for the conformationally flexible MSR protein (Figure 1). In general, the exploration of energy landscapes is integral to conformational sampling mechanisms for many biological Scheme 1. The catalytic and reactivation cycles of MS. During the primary catalytic cycle MS uses enzyme-bound cob(I)alamin to abstract a methyl group from methyl tetrahydrofolate to generate tetrahydrofolate and methylcob(III)alamin. Regeneration of inactivated MS requires one electron derived from NADPH-dependent MSR and methyl transfer from SAM; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine.

Crystal structure and solution characterization of the activation domain of human methionine synthase

Human methionine synthase (hMS) is a multidomain cobalamin‐dependent enzyme that catalyses the conversion of homocysteine to methionine by methyl group transfer. We report here the 1.6 Å crystal structure of the C‐terminal activation domain of hMS. The structure is C‐shaped with the core comprising mixed α and β regions, dominated by a twisted antiparallel β sheet with a β‐meander region. These features, including the positions of the active‐site residues, are similar to the activation domain of Escherichia coli cobalamin‐dependent MS (MetH). Structural and solution studies suggest a small proportion of hMS activation domain exists in a dimeric form, which contrasts with the monomeric form of the E. coli homologue. Fluorescence studies show that human activation domain interacts with the FMN‐binding domain of human methionine synthase reductase (hMSR). This interaction is enhanced in the presence of S‐adenosyl‐methionine. Binding of the D963E/K1071N mutant activation domain to the FMN domain of MSR is weaker than with wild‐type activation domain. This suggests that one or both of the residues D963 and K1071 are important in partner binding. Key differences in the sequences and structures of hMS and MetH activation domains are recognized and include a major reorientation of an extended 310‐containing loop in the human protein. This structural alteration might reflect differences in their respective reactivation complexes and/or potential for dimer formation. The reported structure is a component of the multidomain hMS : MSR complex, and represents an important step in understanding the impact of clinical mutations and polymorphisms in this key electron transfer complex.

Kinetic analysis of cytochrome P450 reductase fromArtemisia annua reveals accelerated rates of NADPH‐dependent flavin reduction

Cytochrome P450 reductase from Artemisia annua (aaCPR) is a diflavin enzyme that has been employed for the microbial synthesis of artemisinic acid (a semi‐synthetic precursor of the anti‐malarial drug, artemisinin) based on its ability to transfer electrons to the cytochrome P450 monooxygenase, CYP71AV1. We have isolated recombinant aaCPR (with the N‐terminal transmembrane motif removed) from Escherichia coli and compared its kinetic and thermodynamic properties with other CPR orthologues, most notably human CPR. The FAD and FMN redox potentials and the macroscopic kinetic constants associated with cytochrome c3+ reduction for aaCPR are comparable to that of other CPR orthologues, with the exception that the apparent binding affinity for the oxidized coenzyme is ~ 30‐fold weaker compared to human CPR. CPR from A. annua shows a 3.5‐fold increase in uncoupled NADPH oxidation compared to human CPR and a strong preference (85 100‐fold) for NADPH over NADH. Strikingly, reduction of the enzyme by the first and second equivalent of NADPH is much faster in aaCPR, with rates of > 500 and 17 s−1 at 6 °C. We also optically detect a charge‐transfer species that rapidly forms in < 3 ms and then persists during the reductive half reaction. Additional stopped‐flow kinetic studies with NADH and (R)‐[4‐2H]NADPH suggest that the accelerated rate of flavin reduction is attributed to the relatively weak binding affinity of aaCPR for NADP+.

Aromatic substitution of the FAD‐shielding tryptophan reveals its differential role in regulating electron flux in methionine synthase reductase and cytochrome P450 reductase

Methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR) transfer reducing equivalents from NADPH via an FAD and FMN cofactor to a redox partner protein. In both enzymes, hydride transfer from NADPH to FAD requires displacement of a conserved tryptophan that lies coplanar to the FAD isoalloxazine ring. Swapping the tryptophan for a smaller aromatic side chain revealed a distinct role for the residue in regulating MSR and CPR catalysis. MSR W697F and W697Y showed enhanced catalysis, noted by increases in kcat and kcat/Km(NADPH) for steady‐state cytochrome c3+ reduction and a 10‐fold increase in the rate constant (kobs1) associated with hydride transfer. Elevated primary kinetic isotope effects on kobs1 for W697F and W697Y suggest that preceding isotopically insensitive steps like displacement of W697 are less rate determining. MSR W697Y, but not MSR W697F, showed detectable formation of the disemiquinone intermediate, indicating that the polarity of the aromatic side chain influences the rate of interflavin electron transfer. By contrast, the CPR variants (W676F and W676Y) displayed modest decreases in cytochrome c3+ reduction, a 30‐ and 3.5‐fold decrease in the rate of FAD reduction, accumulation of a FADH2–NADP+ charge‐transfer complex and dramatically suppressed rates of interflavin electron transfer. We conclude for MSR that hydride transfer is ‘gated’ by the free energy required to disrupt dispersion forces between the FAD isoalloxazine ring and W697. By contrast, the bulky indole ring of W676 accelerates catalysis in CPR by lowering the energy barrier for displacement of the oxidized nicotinamide ring coplanar with the FAD.