Root-secreted nucleosides: signaling chemoattractants of rhizosphere bacteria.

The rhizosphere is a complex ecosystem, consisting of a narrow soil zone influenced by plant roots and inhabited by soil-borne microorganisms. Plants actively shape the rhizosphere microbiome through root exudates. Some metabolites are signaling molecules specifically functioning as chemoattractants rather than nutrients. These elusive signaling molecules have been sought for several decades, and yet little progress has been made. Root-secreted nucleosides and deoxynucleosides were detected in exudates of various plants by targeted ultra-performance liquid chromatography-mass spectrometry/mass spectrometry. Rhizobacteria were isolated from the roots of Helianthemum sessiliflorum carrying the mycorrhizal desert truffle Terfezia boudieri. Chemotaxis was determined by a glass capillary assay or plate assays on semisolid agar and through a soil plate assay. Nucleosides were identified in root exudates of plants that inhabit diverse ecological niches. Nucleosides induced positive chemotaxis in plant beneficial bacteria Bacillus pumilus, Bacillus subtilis, Pseudomonas turukhanskensis spp., Serratia marcescens, and the pathogenic rhizobacterium Xanthomonas campestris and E coli. In a soil plate assay, nucleosides diffused to substantial distances and evoked chemotaxis under conditions as close as possible to natural environments. This study implies that root-secreted nucleosides are involved in the assembly of the rhizosphere bacterial community by inducing chemotaxis toward plant roots. In animals, nucleoside secretion known as "purinergic signaling" is involved in communication between cells, physiological processes, diseases, phagocytic cell migration, and bacterial activity. The coliform bacterium E. coli that inhabits the lower intestine of warm-blooded organisms also attracted to nucleosides, implying that nucleosides may serve as a common signal for bacterial species inhabiting distinct habitats. Taken together, all these may indicate that chemotaxis signaling by nucleosides is a conserved universal mechanism that encompasses living kingdoms and environments and should be given further attention in plant rhizosphere microbiome research.

The Microbiome Structure of the Symbiosis between the Desert Truffle Terfezia boudieri and Its Host Plant Helianthemum sessiliflorum.

The desert truffle Terfezia boudieri is an ascomycete fungus that forms ect-endomycorrhiza in the roots of plants belonging to Cistaceae. The fungus forms hypogeous edible fruit bodies, appreciated as gourmet food. Truffles and host plants are colonized by various microbes, which may contribute to their development. However, the diversity and composition of the bacterial community under field conditions in the Negev desert are still unknown. The overall goal of this research was to identify the rhizosphere microbial community supporting the establishment of a symbiotic association between T. boudieri and Helianthemum sessiliflorum. The bacterial community was characterized by fruiting bodies, mycorrhized roots, and rhizosphere soil. Based on next-generation sequencing meta-analyses of the 16S rRNA gene, we discovered diverse bacterial communities of fruit bodies that differed from those found in the roots and rhizosphere. Families of Proteobacteria, Planctomycetes, and Actinobacteria were present in all four samples. Alpha diversity analysis revealed that the rhizosphere and roots contain significantly higher bacterial species numbers compared to the fruit. Additionally, ANOSIM and PCoA provided a comparative analysis of the bacterial taxa associated with fruiting bodies, roots, and rhizosphere. The core microbiome described consists of groups whose biological role triggers important traits supporting plant growth and fruit body development.

Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri.

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 µM of acetosyringone, attaining a bacterial optical density of 0.3 OD600; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.

Characterization of Morphology, Volatile Profiles, and Molecular Markers in Edible Desert Truffles from the Negev Desert.

Desert truffles are mycorrhizal, hypogeous fungi considered a delicacy. On the basis of morphological characters, we identified three desert truffle species that grow in the same habitat in the Negev desert. These include Picoa lefebvrei (Pat.), Tirmania nivea (Desf.) Trappe, and Terfezia boudieri (Chatain), all associated with Helianthemum sessiliflorum. Their taxonomy was confirmed by PCR-RFLP. The main volatiles of fruit bodies of T. boudieri and T. nivea were 1-octen-3-ol and hexanal; however, volatiles of the latter species further included branched-chain amino acid derivatives such as 2-methylbutanal and 3-methylbutanal, phenylalanine derivatives such as benzaldehyde and benzenacetaldehyde, and methionine derivatives such as methional and dimethyl disulfide. The least aromatic truffle, P. lefebvrei, contained low levels of 1-octen-3-ol as the main volatile. Axenic mycelia cultures of T. boudieri displayed a simpler volatile profile compared to its fruit bodies. This work highlights differences in the volatile profiles of desert truffles and could hence be of interest for selecting and cultivating genotypes with the most likable aroma.

Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate.

Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1' atom of ThDP (replaced by Val51) or any other potential acid-base side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0-6.2 (pK(a) of 6.2) for TSA formation from pH 7.0-7.7 in wild-type GCL. This pK(a) is similar to the pK(a) of 6.1 for the 1',4'-iminopyrimidine (IP)-4'-aminopyrimidinium (APH(+)) protonic equilibrium, suggesting that the same groups control both ThDP protonation and TSA formation. The key covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center variants with substitution at I393 could synthesize (S)-acetolactate from pyruvate solely, and acetylglycolate derived from pyruvate as the acetyl donor and glyoxylate as the acceptor, implying that this substitutent favored pyruvate as the donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the predecarboxylation intermediate analogues derived from acetylphosphinate, propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be catalyzed by ThDP itself, once the APH(+) form is accessible.

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.

Allosteric regulation in Acetohydroxyacid Synthases (AHASs)--different structures and kinetic behavior in isozymes in the same organisms.

Acetohydroxyacid Synthases (AHASs) have separate small regulatory subunits which specifically activate the catalytic subunits with which they are associated. The binding sites for the inhibitory amino acid(s) (valine or leucine) are in the interface between two ACT (small ligand binding) domains, and are apparently found in all AHAS regulatory subunits. However, the structures and the kinetic mechanisms of the different enzymes are very heterogeneous. Among the three isozymes encoded in the enterobacteria, the regulatory patterns are different, and their different responses to the inhibitory end product valine can be rationalized, at least in part, on the basis of the regulatory subunit structures and differences in catalytic mechanisms. The regulatory subunits in "typical" single AHASs found in other bacteria are similar to that of Escherichia coli isozyme AHAS III. Eukaryotic AHASs have more complex regulatory mechanisms and larger regulatory subunits. Such AHASs have two separate ACT sequence domains on the same regulatory polypeptide and can simultaneously bind two amino acids with synergistic effects. Yeast and fungal AHASs have ATP-binding sequence inserts in their regulatory subunits and are activated by MgATP in addition to being inhibited by valine.

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.

A new Thraustochytrid, strain Fng1, isolated from the surface mucus of the hermatypic coral Fungia granulosa.

Recent evidence suggests that there is a dynamic microbial biota living on the surface and in the mucus layer of many hermatypic coral species that plays an essential role in coral well-being. Most of the studies published to date emphasize the importance of prokaryotic communities associated with the coral mucus in coral health and disease. In this study, we report the presence of a protist (Fng1) in the mucus of the hermatypic coral Fungia granulosa from the Gulf of Eilat. This protist was identified morphologically and molecularly as belonging to the family Thraustochytridae (phylum Stramenopile, order Labyrinthulida), a group of heterotrophs widely distributed in the marine environment. Morphological examination of this strain revealed a nonmotile organism c. 35 mum in diameter, which is able to thrive on carbon-deprived media, and whose growth and morphology are inoculum dependent. Its fatty acid production profile revealed an array of polyunsaturated fatty acids. A similar protist was also isolated from the mucus of the coral Favia sp. In light of these findings, its possible contribution to the coral holobiont is discussed.

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.

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.