Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H2O2 via cysteine-dependent redox short-circuit.

The Cu/Zn-superoxide dismutase (SOD1) is a ubiquitous enzyme that catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide. In addition to this principal reaction, the enzyme is known to catalyze, with various efficiencies, several redox side-reactions using alternative substrates, including biological thiols, all involving the catalytic copper in the enzyme's active-site, which is relatively surface exposed. The accessibility and reactivity of the catalytic copper is known to increase upon SOD1 misfolding, structural alterations caused by a mutation or environmental stresses. These competing side-reactions can lead to the formation of particularly toxic ROS, which have been proposed to contribute to oxidative damage in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects motor neurons. Here, we demonstrated that metal-saturated SOD1WT (holo-SOD1WT) and a familial ALS (fALS) catalytically active SOD1 mutant, SOD1G93A, are capable, under defined metabolic circumstances, to generate cytotoxic quantities of H2O2 through cysteine (CSH)/glutathione (GSH) redox short-circuit. Such activity may drain GSH stores, therefore discharging cellular antioxidant potential. By analyzing the distribution of thiol compounds throughout the CNS, the location of potential hot-spots of ROS production can be deduced. These hot-spots may constitute the origin of oxidative damage to neurons in ALS.

Construction of Structural Mimetics of the Thyrotropin Receptor Intracellular Domain.

The signaling of a G protein-coupled receptor (GPCR) is dictated by the complementary responsiveness of interacting intracellular effectors such as G proteins. Many GPCRs are known to couple to more than one G protein subtype and induce a multitude of signaling pathways, although the in vivo relevance of particular pathways is mostly unrecognized. Dissecting GPCR signaling in terms of the pathways that are activated will boost our understanding of the molecular fundamentals of hormone action. The structural determinants governing the selectivity of GPCR/G protein coupling, however, remain obscure. Here, we describe the design of soluble GPCR mimetics to study the details of the interplay between G-proteins and activators. We constructed functional mimetics of the intracellular domain of a model GPCR, the thyrotropin receptor. We based the construction on a unique scaffold, 6-Helix, an artificial protein that was derived from the elements of the trimer-of-hairpins structure of HIV gp41 and represents a bundle of six α-helices. The 6-Helix scaffold, which endowed the substituted thyrotropin receptor intracellular domain elements with spatial constraints analogous to those found in native receptors, enabled the reconstitution of a microdomain that consists of intracellular loops 2 and 3, and is capable of binding and activating Gα-(s). The 6-Helix-based mimetics could be used as a platform to study the molecular basis of GPCR/G protein recognition. Such knowledge could help investigators develop novel therapeutic strategies for GPCR-related disorders by targeting the GPCR/G protein interfaces and counteracting cellular dysfunctions via focused tuning of GPCR signaling.

Superoxide Dismutase 1 (SOD1)-Derived Peptide Inhibits Amyloid Aggregation of Familial Amyotrophic Lateral Sclerosis SOD1 Mutants.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the death of the upper and lower motor neurons. Superoxide dismutase 1 (SOD1) is an ALS pathogenic protein, whose misfolding results in the formation of amyloid aggregates. The mechanism underlying SOD1 pathogenesis in ALS remains obscure, but one possible mechanism involves gain-of-interaction, in which the misfolded soluble SOD1 forms abnormal protein-protein interactions (PPIs) with various cellular proteins, including with other SOD1 molecules, thereby interfering with their function. The structural basis of this gain-of-interaction mechanism is unknown. Here, we characterized the backbone dynamics landscape of misfolded SOD1 to pinpoint surface areas predisposed to aberrant PPIs. This analysis enabled us to formulate a working hypothesis for the mechanism of the gain-of-function of misfolded SOD1, according to which an abnormal PPI potential results from the increased mobility of the SOD1 surface backbone. Guided by the backbone dynamics landscape, we have identified a SOD1-derived peptide that can bind SOD1 proteins and divert the typical amyloid aggregation of ALS-related SOD1 mutants toward a potentially less toxic amorphous aggregation pathway.

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes.

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.

Tethered ribozyme ligation enables detection of molecular proximity in homogeneous solutions.

In contemporary drug discovery, bulk selection represents an important alternative to time consuming and expensive high-throughput screening. The selection methods, however, generally rely on affinity separation, a step that limits overall selection process efficiency. To overcome common drawbacks of conventional methods, we exploited the unique catalytic properties of an artificial enzyme, ribozyme ligase, to develop a selection methodology in which the entire detection process takes place in a homogeneous solution, thus eliminating the need for affinity separation. A molecular target is associated with the ribozyme, and library compounds are attached to a barcoded oligonucleotide that is a substrate for the ribozyme ligase. Spatial proximity resulting from specific target-compound interactions increases the probability of ribozyme ligation to the oligo-substrate, thus differentiating the interacting species from the bulk mixture. The covalent link formed between the ribozyme and target-interacting compounds diminishes the mass-action effect on the efficiency with which low-affinity and rare active species are detected. In addition, the magnitude of the detection signal associated with the interaction event renders the methodology an efficient platform for identifying inhibitors of intermolecular interactions. The proposed solution-based tethered ribozyme-ligation proximity detection method may facilitate the discovery of target-interacting compounds using both library selection and high-throughput screening approaches.

Cloning, mutagenesis, and characterization of the microalga Parietochloris incisa acetohydroxyacid synthase, and its possible use as an endogenous selection marker.

Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic-resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched-chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the wild-type and mutant proteins were characterized in the AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wild-type and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild-type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type.

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate-thiamin diphosphate adduct in AHAS I.

Acetohydroxy acid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate (ThDP)-dependent decarboxylase-ligase that catalyzes the first common step in the biosynthesis of branched-chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl-ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing-quench NMR method or stopped-flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2-lactyl-ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2-KB, putting into question whether AHAS operates via a pure ping-pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor-acceptor:enzyme complex followed by covalent, ThDP-promoted catalysis with concerted decarboxylation-carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS.

Significant catalytic roles for Glu47 and Gln 110 in all four of the C-C bond-making and -breaking steps of the reactions of acetohydroxyacid synthase II.

Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the first common step in the biosynthesis of branched-chain amino acids, condensation of pyruvate with a second 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme II from Escherichia coli is specific for pyruvate as the first donor substrate but exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. In previous studies relying on steady state and transient kinetics, substrate competition and detailed analysis of the distribution of intermediates in the steady-state, we have identified several residues which confer specificity for the donor and acceptor substrates, respectively. Here, we examine the roles of active site polar residues Glu47, Gln110, Lys159, and His251 for elementary steps of catalysis using similar approaches. While Glu47, the conserved essential glutamate conserved in all ThDP-dependent enzymes whose carboxylate is in H-bonding distance of the ThDP iminopyrimidine N1', is involved as expected in cofactor activation, substrate binding, and product elimination, our studies further suggest a crucial catalytic role for it in the carboligation of the acceptor and the hydroxyethyl-ThDP enamine intermediate. The Glu47-cofactor proton shuttle acts in concert with Gln110 in the carboligation. We suggest that either the transient oxyanion on the acceptor carbonyl is stabilized by H-bonding to the glutamine side chain, or carboligation involves glutamine tautomerization and the elementary reactions of addition and protonation occur in a concerted manner. This is in contrast to the situation in other ThDP enzymes that catalyze a carboligation, such as, e.g., transketolase or benzaldehyde lyase, where histidines act as general acid/base catalysts. Our studies further suggest global catalytic roles for Gln110 and Glu47, which are engaged in all major bond-breaking and bond-making steps. In contrast to earlier suggestions, Lys159 has a minor effect on the kinetics and specificity of AHAS II, far less than does Arg276, previously shown to influence the specificity for a 2-ketoacid as a second substrate. His251 has a large effect on donor substrate binding, but this effect masks any other effects of replacement of His251.

Role of the C-terminal domain of the regulatory subunit of AHAS isozyme III: use of random mutagenesis with in vivo reconstitution (REM-ivrs).

In order to clarify the role of the C-terminal domain of the ilvH protein (the regulatory subunit of enterobacterial AHAS isozyme III, whose structure has been solved and reported by Kaplun et al., J Mol Biol 357, 951, 2006) in the process of valine inhibition of AHAS III, we developed a procedure that randomly mutagenizes a specific segment of a gene through error-prone PCR and screens for mutants on the basis of the properties of the holoenzymes reconstituted in vivo (REM-ivrs). Previous work showed that the N-terminal domain includes the valine-binding ACT domain of the regulatory subunit and is sufficient to completely activate the catalytic subunit, but that this domain cannot confer valine sensitivity on the reconstituted enzyme. It appeared that the C-terminal domain of the ilvH is involved in some way in "signal transmission" of the inhibition by valine. As knowledge of the structure of AHAS holoenzymes and the interactions between the catalytic and regulatory subunits is very limited, a procedure that focuses on the C-terminal domain in the ilvH gene could add to the understanding of the mechanism by which the binding of valine to the regulatory subunit is coupled to inhibition of the catalytic activity. In the REM-ivrs procedure, a medium copy (~40 copies) plasmid expressing ilvH with a Val(r) mutation confers the Val(r) phenotype upon bacteria. All the single missense mutations produced by REM-ivrs were found to be localized to the interface between the C-terminal domains of two monomers in the ilvH dimer. The loss of specific contacts involved in inter-monomer interactions in this region might conceivably disrupt the structure of the C-terminal domain itself. Biochemical study of an isolated Val(r) mutant elicited by the REM-ivrs method detected no binding of radioactively labeled valine, as previously found in a truncation mutant. The idea that the C-terminal domain has a specific "signal-transmission" role was also contradicted by examination of the thermal stability of the Val(r) REM-ivrs variants by the Thermofluor method, which does not detect any signs of biphasic melting behavior for any of the mutants. We propose that the mutants of ilvH isolated by the REM-ivrs method differ from the wild-type in the equilibrium between two states of the enzyme. Without the specific interdomain contacts of the wild-type ilvH protein, the holoenzyme reconstituted from mutant regulatory subunits is apparently in a state with uninhibited activity and low affinity for valine.

Valine 375 and phenylalanine 109 confer affinity and specificity for pyruvate as donor substrate in acetohydroxy acid synthase isozyme II from Escherichia coli.

Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate-dependent enzyme that catalyzes the condensation of pyruvate with either another pyruvate molecule (product acetolactate) or 2-ketobutyrate (product acetohydroxybutyrate) as the first common step in the biosynthesis of branched-chain amino acids in plants, bacteria, algae, and fungi. AHAS isozyme II from Escherichia coli exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as acceptor, which was shown to result from a stronger hydrophobic interaction of the ethyl substituent of 2-KB with the side chain of Trp464 in multiple, apparently committed steps of catalysis. Here, we have elucidated the molecular determinants conferring specificity for pyruvate as the sole physiological donor substrate. Structural studies and sequence alignments of the POX subfamily of ThDP enzymes that act on pyruvate indicate that a valine and a phenylalanine hydrophobically interact with the methyl substituent of pyruvate. Kinetic and thermodynamic studies on AHAS isozyme II variants with substitutions at these positions (Val375Ala, Val375Ile, and Phe109Met) were carried out. While Val375 variants exhibit a slightly reduced k(cat) with a moderate increase of the apparent K(M) of pyruvate, both substrate affinity and k(cat) are significantly compromised in AHAS Phe109Met. The specificity for 2-ketobutyrate as acceptor is not altered in the variants. Binding of acylphosphonates as analogues of donor substrates was analyzed by circular dichroism spectroscopy and stopped-flow kinetics. While binding of the pyruvate analogue is 10-100-fold compromised in all variants, Val375Ala binds the 2-KB analogue better than the wild type and with higher affinity than the pyruvate analogue, suggesting steric constraints imposed by Val375 as a major determinant for the thermodynamically favored binding of pyruvate in AHAS. NMR-based intermediate analysis at steady state reveals that a mutation of either Val375 or Phe109 is detrimental for unimolecular catalytic steps in which tetrahedral intermediates are involved, such as substrate addition to the cofactor and product liberation. This observation implies Val375 and Phe109 to not only conjointly mediate substrate binding and specificity but moreover to ensure a proper orientation of the donor substrate and intermediates for correct orbital alignment in multiple transition states.

Interactions between large and small subunits of different acetohydroxyacid synthase isozymes of Escherichia coli.

The large, catalytic subunits (LSUs; ilvB, ilvG and ilvI, respectively) of enterobacterial acetohydroxyacid synthases isozymes (AHAS I, II and III) have molecular weights approximately 60 kDa and are paralogous with a family of other thiamin diphosphate dependent enzymes. The small, regulatory subunits (SSUs) of AHAS I and AHAS III (ilvN and ilvH) are required for valine inhibition, but ilvN and ilvH can only confer valine sensitivity on their own LSUs. AHAS II is valine resistant. The LSUs have only approximately 15, <<1 and approximately 3%, respectively, of the activity of their respective holoenzymes, but the holoenzymes can be reconstituted with complete recovery of activity. We have examined the activation of each of the LSUs by SSUs from different isozymes and ask to what extent such activation is specific; that is, is effective nonspecific interaction possible between LSUs and SSUs of different isozymes? To our surprise, the AHAS II SSU ilvM is able to activate the LSUs of all three of the isozymes, and the truncated AHAS III SSUs ilvH-Delta80, ilvH-Delta86 and ilvH-Delta89 are able to activate the LSUs of both AHAS I and AHAS III. However, none of the heterologously activated enzymes have any feedback sensitivity. Our results imply the existence of a common region in all three LSUs to which regulatory subunits may bind, as well as a similarity between the surfaces of ilvM and the other SSUs. This surface must be included within the N-terminal betaalphabetabetaalphabeta-domain of the SSUs, probably on the helical face of this domain. We suggest hypotheses for the mechanism of valine inhibition, and reject one involving induced dissociation of subunits.

Allosteric regulation of Bacillus subtilis threonine deaminase, a biosynthetic threonine deaminase with a single regulatory domain.

The enzyme threonine deaminase (TD) is a key regulatory enzyme in the pathway for the biosynthesis of isoleucine. TD is inhibited by its end product, isoleucine, and this effect is countered by valine, the product of a competing biosynthetic pathway. Sequence and structure analyses have revealed that the protomers of many TDs have C-terminal regulatory domains, composed of two ACT-like subdomains, which bind isoleucine and valine, while others have regulatory domains of approximately half the length, composed of only a single ACT-like domain. The regulatory responses of TDs from both long and short sequence varieties appear to have many similarities, but there are significant differences. We describe here the allosteric properties of Bacillus subtilis TD ( bsTD), which belongs to the short variety of TD sequences. We also examine the effects of several mutations in the regulatory domain on the kinetics of the enzyme and its response to effectors. The behavior of bsTD can be analyzed and rationalized using a modified Monod-Wyman-Changeux model. This analysis suggests that isoleucine is a negative effector, and valine is a very weak positive effector, but that at high concentrations valine inhibits activity by competing with threonine for binding to the active site. The behavior of bsTD is contrasted with the allosteric behavior reported for TDs from Escherichia coli and Arabidopsis thaliana, TDs with two subdomains. We suggest a possible evolutionary pathway to the more complex regulatory effects of valine on the activity of TDs of the long sequence variety, e.g., E. coli TD.

Glyoxylate carboligase lacks the canonical active site glutamate of thiamine-dependent enzymes.

Thiamine diphosphate (ThDP), a derivative of vitamin B1, is an enzymatic cofactor whose special chemical properties allow it to play critical mechanistic roles in a number of essential metabolic enzymes. It has been assumed that all ThDP-dependent enzymes exploit a polar interaction between a strictly conserved glutamate and the N1' of the ThDP moiety. The crystal structure of glyoxylate carboligase challenges this paradigm by revealing that valine replaces the conserved glutamate. Through kinetic, spectroscopic and site-directed mutagenesis studies, we show that although this extreme change lowers the rate of the initial step of the enzymatic reaction, it ensures efficient progress through subsequent steps. Glyoxylate carboligase thus provides a unique illustration of the fine tuning between catalytic stages imposed during evolution on enzymes catalyzing multistep processes.

Construction of an active acetohydroxyacid synthase I with a flexible linker connecting the catalytic and the regulatory subunits.

Acetohydroxyacid synthase I (AHAS I), one of three isozymes in Escherichia coli catalyzing the first common step in the biosynthesis of branched amino acids, is composed of two kinds of subunits. The large catalytic (B) and small regulatory (N) subunits of the holoenzyme dissociate and associate freely and rapidly and are quite different in size, charge and hydrophobicity, so that high resolution purification methods lead to partial separation of subunits and to heterogeneity. We have prepared several linked AHAS I proteins, in which the large subunit B with a hexahistidine-tag at the N-terminus, was covalently joined by a flexible linker, containing several (X) amino acids, to the small subunit N to form His6-BuXN polypeptides. All linked BuXN polypeptides have similar specific activity, sensitivity to valine and substrate specificity as the wild type holoenzyme. The most successful BuXN linked protein (Bu30N-r) was inserted into and expressed in yeast and its catalytic properties were tested.

Structure of the regulatory subunit of acetohydroxyacid synthase isozyme III from Escherichia coli.

The enzyme acetohydroxyacid synthase (AHAS) catalyses the first common step in the biosynthesis of the three branched-chain amino acids. Enzymes in the AHAS family generally consist of regulatory and catalytic subunits. Here, we describe the first crystal structure of an AHAS regulatory subunit, the ilvH polypeptide, determined at a resolution of 1.75 A. IlvH is the regulatory subunit of one of three AHAS isozymes expressed in Escherichia coli, AHAS III. The protein is a dimer, with two beta alpha beta beta alpha beta ferredoxin domains in each monomer. The two N-terminal domains assemble to form an ACT domain structure remarkably close to the one predicted by us on the basis of the regulatory domain of 3-phosphoglycerate dehydrogenase (3PGDH). The two C-terminal domains combine so that their beta-sheets are roughly positioned back-to-back and perpendicular to the extended beta-sheet of the N-terminal ACT domain. On the basis of the properties of mutants and a comparison with 3PGDH, the effector (valine) binding sites can be located tentatively in two symmetrically related positions in the interface between a pair of N-terminal domains. The properties of mutants of the ilvH polypeptide outside the putative effector-binding site provide further insight into the functioning of the holoenzyme. The results of this study open avenues for further studies aimed at understanding the mechanism of regulation of AHAS by small-molecule effectors.

Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties.

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of "typical" AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.

Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy.

Acetohydroxy acid synthase (AHAS) and related enzymes catalyze the production of chiral compounds [(S)-acetolactate, (S)-acetohydroxybutyrate, or (R)-phenylacetylcarbinol] from achiral substrates (pyruvate, 2-ketobutyrate, or benzaldehyde). The common methods for the determination of AHAS activity have shortcomings. The colorimetric method for detection of acyloins formed from the products is tedious and does not allow time-resolved measurements. The continuous assay for consumption of pyruvate based on its absorbance at 333 nm, though convenient, is limited by the extremely small extinction coefficient of pyruvate, which results in a low signal-to-noise ratio and sensitivity to interfering absorbing compounds. Here, we report the use of circular dichroism spectroscopy for monitoring AHAS activity. This method, which exploits the optical activity of reaction products, displays a high signal-to-noise ratio and is easy to perform both in time-resolved and in commercial modes. In addition to AHAS, we examined the determination of activity of glyoxylate carboligase. This enzyme catalyzes the condensation of two molecules of glyoxylate to chiral tartronic acid semialdehyde. The use of circular dichroism also identifies the product of glyoxylate carboligase as being in the (R) configuration.

Column flow reactor using acetohydroxyacid synthase I from Escherichia coli as catalyst in continuous synthesis of R-phenylacetyl carbinol.

We tested the possibility of utilizing acetohydroxyacid synthase I (AHAS I) from Escherichia coli in a continuous flow reactor for production of R-phenylacetyl carbinol (R-PAC). We constructed a fusion of the large, catalytic subunit of AHAS I with a cellulose binding domain (CBD). This allowed purification of the enzyme and its immobilization on cellulose in a single step. After immobilization, AHAS I is fully active and can be used as a catalyst in an R-PAC production unit, operating either in batch or continuous mode. We propose a simplified mechanistic model that can predict the product output of the AHAS I-catalyzed reaction. This model should be useful for optimization and scaling up of a R-PAC production unit, as demonstrated by a column flow reactor.

The carboligation reaction of acetohydroxyacid synthase II: steady-state intermediate distributions in wild type and mutants by NMR.

The thiamin diphosphate (ThDP)-dependent enzyme acetohydroxyacid synthase (AHAS) catalyzes the first common step in branched-chain amino acid biosynthesis. By specific ligation of pyruvate with the alternative acceptor substrates 2-ketobutyrate and pyruvate, AHAS controls the flux through this branch point and determines the relative rates of synthesis of isoleucine, valine, and leucine, respectively. We used detailed NMR analysis to determine microscopic rate constants for elementary steps in the reactions of AHAS II and mutants altered at conserved residues Arg-276, Trp-464, and Met-250. In Arg276Lys, both the condensation of the enzyme-bound hydroxyethyl-ThDP carbanion/enamine (HEThDP) with the acceptor substrates and acetohydroxyacid release are slowed several orders of magnitude relative to the wild-type enzyme. We propose that the interaction of the guanidinium moiety of Arg-264 with the carboxylate of the acceptor ketoacid provides an optimal alignment of substrate and HEThDP orbitals in the reaction trajectory for acceptor ligation, whereas its interaction with the carboxylate of the covalent HEThDP-acceptor adduct plays a similar role in product release. Both Trp-464 and Met-250 affect the acceptor specificity. The high preference for ketobutyrate in the wild-type enzyme is lost in Trp464Leu as a consequence of similar forward rate constants of carboligation and product release for the alternative acceptors. In Met250Ala, the turnover rate is determined by the condensation of HEThDP with pyruvate and release of the acetolactate product, whereas the parallel steps with 2-ketobutyrate are considerably faster. We speculate that the specificity of carboligation and product liberation may be cumulative if the former is not completely committed.

Substrate range of acetohydroxy acid synthase I from Escherichia coli in the stereoselective synthesis of alpha-hydroxy ketones.

Acetohydroxy acid synthase I appears to be the most effective of the AHAS isozymes found in Escherichia coli in the chiral synthesis of phenylacetyl carbinol from pyruvate and benzaldehyde. We report here the exploration of a range of aldehydes as substrates for AHAS I and demonstrate that the enzyme can accept a wide variety of substituted benzaldehydes, as well as heterocyclic and heteroatomic aromatic aldehydes, to produce chiral carbinols. The active site of AHAS I does not appear to impose serious steric constraints on the acceptor substrate. The influence of electronic effects on the reaction has been probed using substituted benzaldehydes as substrates. The electrophilicity of the aldehyde acceptor substrates is most important to their reactivity, but the lipophilicity of substituents also affects their reactivity. AHAS I is an effective biosynthetic platform for production of a variety of alpha-hydroxy ketones, compounds with considerable potential as pharmacological precursors.