Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

The Sinorhizobium meliloti NspS-MbaA system affects biofilm formation, exopolysaccharide production and motility in response to specific polyamines.

We previously showed that specific polyamines (PAs) present in the extracellular environment markedly affect extracellular polysaccharide (EPS) production, biofilm formation and motility in Sinorhizobium meliloti Rm8530. We hypothesized that extracellular PA signals were sensed and transduced by the NspS and MbaA proteins, respectively, which are homologs of the PA-sensing, c-di-GMP modulating NspS-MbaA proteins described in Vibrio cholerae. Here we show that the decrease in biofilm formation and EPS production in the quorum-sensing (QS)-deficient S. meliloti wild-type strain 1021 in cultures containing putrescine or spermine did not occur in a 1021 nspS mutant (1021 nspS). The transcriptional expression of nspS in strain 1021 was significantly increased in cultures containing either of these polyamines, but not by exogenous cadaverine, 1,3-diaminopropane (DAP), spermidine (Spd) or norspermidine (NSpd). Cell aggregation in liquid cultures did not differ markedly between strain 1021 and 1021 nspS in the presence or absence of PAs. The S. meliloti QS-proficient Rm8530 wild-type and nspS mutant (Rm8530 nspS) produced similar levels of biofilm under control conditions and 3.2- and 2.2-fold more biofilm, respectively, in cultures with NSpd, but these changes did not correlate with EPS production. Cells of Rm8530 nspS aggregated from two- to several-fold more than the wild-type in cultures without PAs or in those containing Spm. NSpd, Spd and DAP differently affected swimming and swarming motility in strains 1021 and Rm8530 and their respective nspS mutants. nspS transcription in strain Rm8530 was greatly reduced by exogenous Spm. Bioinformatic analysis revealed similar secondary structures and functional domains in the MbaA proteins of S. meliloti and V. cholerae, while their NspS proteins differed in some residues implicated in polyamine recognition in the latter species. NspS-MbaA homologs occur in a small subset of soil and aquatic bacterial species that commonly interact with eukaryotes. We speculate that the S. meliloti NspS-MbaA system modulates biofilm formation, EPS production and motility in response to environmental or host plant-produced PAs.

A Tar aspartate receptor and Rubisco-like protein substitute biotin in the growth of rhizobial strains.

Biotin is a key cofactor of metabolic carboxylases, although many rhizobial strains are biotin auxotrophs. When some of these strains were serially subcultured in minimal medium, they showed diminished growth and increased excretion of metabolites. The addition of biotin, or genetic complementation with biotin synthesis genes resulted in full growth of Rhizobium etli CFN42 and Rhizobium phaseoli CIAT652 strains. Half of rhizobial genomes did not show genes for biotin biosynthesis, but three-quarters had genes for biotin transport. Some strains had genes for an avidin homologue (rhizavidin), a protein with high affinity for biotin but an unknown role in bacteria. A CFN42-derived rhizavidin mutant showed a sharper growth decrease in subcultures, revealing a role in biotin storage. In the search of biotin-independent growth of subcultures, CFN42 and CIAT652 strains with excess aeration showed optimal growth, as they also did, unexpectedly, with the addition of aspartic acid analogues α- and N-methyl aspartate. Aspartate analogues can be sensed by the chemotaxis aspartate receptor Tar. A tar homologue was identified and its mutants showed no growth recovery with aspartate analogues, indicating requirement of the Tar receptor in such a phenotype. Additionally, tar mutants did not recover full growth with excess aeration. A Rubisco-like protein was found to be necessary for growth as the corresponding mutants showed no recovery either with high aeration or aspartate analogues; also, diminished carboxylation was observed. Taken together, our results indicate a route of biotin-independent growth in rhizobial strains that included oxygen, a Tar receptor and a previously uncharacterized Rubisco-like protein.

Mutation of ßGln114 to Ala Alters the Stabilities of Allosteric States in Tryptophan Synthase Catalysis.

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5'-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and ß-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3'-phosphate and l-Ser to l-Trp. In this process, the roles played by ß-site residues proximal to the PLP cofactor have not yet been fully established. ßGln114 is one such residue. To explore the roles played by ßQ114, we conducted a detailed investigation of the ßQ114A mutation on the structure and function of tryptophan synthase. Initial steady-state kinetic and static ultraviolet-visible spectroscopic analyses showed the Q to A mutation impairs catalytic activity and alters the stabilities of intermediates in the ß-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and ßQ114A mutant enzymes. These comparisons establish that the protein structural changes are limited to the Gln to Ala replacement, the loss of hydrogen bonds among the side chains of ßGln114, ßAsn145, and ßArg148, and the inclusion of waters in the cavity created by substitution of the smaller Ala side chain. Because the conformations of the open and closed allosteric states are not changed by the mutation, we hypothesize that the altered properties arise from the lost hydrogen bonds that alter the relative stabilities of the open (ßT state) and closed (ßR state) conformations of the ß-subunit and consequently alter the distribution of intermediates along the ß-subunit catalytic path.

Backbone assignments and conformational dynamics in the S. typhimurium tryptophan synthase α-subunit from solution-state NMR.

Backbone assignments for the isolated α-subunit of Salmonella typhimurium tryptophan synthase (TS) are reported based on triple resonance solution-state NMR experiments on a uniformly 2H,13C,15N-labeled sample. From the backbone chemical shifts, secondary structure and random coil index order parameters (RCI-S2) are predicted. Titration with the 3-indole-D-glycerol 3'-phosphate analog, N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), leads to chemical shift perturbations indicative of conformational changes from which an estimate of the dissociation constant is obtained. Comparisons of the backbone chemical-shifts, RCI-S2 values, and site-specific relaxation times with and without F9 reveal allosteric changes including modulation in secondary structures and loop rigidity induced upon ligand binding. A comparison is made to the X-ray crystal structure of the α-subunit in the full TS αßßα bi-enzyme complex and to two new X-ray crystal structures of the isolated TS α-subunit reported in this work.

Polyamines produced by Sinorhizobium meliloti Rm8530 contribute to symbiotically relevant phenotypes ex planta and to nodulation efficiency on alfalfa.

In nitrogen-fixing rhizobia, emerging evidence shows significant roles for polyamines in growth and abiotic stress resistance. In this work we show that a polyamine-deficient ornithine decarboxylase null mutant (odc2) derived from Sinorhizobium meliloti Rm8530 had significant phenotypic differences from the wild-type, including greatly reduced production of exopolysaccharides (EPS; ostensibly both succinoglycan and galactoglucan), increased sensitivity to oxidative stress and decreased swimming motility. The introduction of the odc2 gene borne on a plasmid into the odc2 mutant restored wild-type phenotypes for EPS production, growth under oxidative stress and swimming. The production of calcofluor-binding EPS (succinoglycan) by the odc2 mutant was also completely or mostly restored in the presence of exogenous spermidine (Spd), norspermidine (NSpd) or spermine (Spm). The odc2 mutant formed about 25 % more biofilm than the wild-type, and its ability to form biofilm was significantly inhibited by exogenous Spd, NSpd or Spm. The odc2 mutant formed a less efficient symbiosis with alfalfa, resulting in plants with significantly less biomass and height, more nodules but less nodule biomass, and 25 % less nitrogen-fixing activity. Exogenously supplied Put was not able to revert these phenotypes and caused a similar increase in plant height and dry weight in uninoculated plants and in those inoculated with the wild-type or odc2 mutant. We discuss ways in which polyamines might affect the phenotypes of the odc2 mutant.

Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti.

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti, that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

Proteins in the periplasmic space and outer membrane vesicles of Rhizobium etli CE3 grown in minimal medium are largely distinct and change with growth phase.

Rhizobium etli CE3 grown in succinate-ammonium minimal medium (MM) excreted outer membrane vesicles (OMVs) with diameters of 40 to 100 nm. Proteins from the OMVs and the periplasmic space were isolated from 6 and 24 h cultures and identified by proteome analysis. A total of 770 proteins were identified: 73.8 and 21.3 % of these occurred only in the periplasm and OMVs, respectively, and only 4.9 % were found in both locations. The majority of proteins found in either location were present only at 6 or 24 h: in the periplasm and OMVs, only 24 and 9 % of proteins, respectively, were present at both sampling times, indicating a time-dependent differential sorting of proteins into the two compartments. The OMVs contained proteins with physiologically varied roles, including Rhizobium adhering proteins (Rap), polysaccharidases, polysaccharide export proteins, auto-aggregation and adherence proteins, glycosyl transferases, peptidoglycan binding and cross-linking enzymes, potential cell wall-modifying enzymes, porins, multidrug efflux RND family proteins, ABC transporter proteins and heat shock proteins. As expected, proteins with known periplasmic localizations (phosphatases, phosphodiesterases, pyrophosphatases) were found only in the periplasm, along with numerous proteins involved in amino acid and carbohydrate metabolism and transport. Nearly one-quarter of the proteins present in the OMVs were also found in our previous analysis of the R. etli total exproteome of MM-grown cells, indicating that these nanoparticles are an important mechanism for protein excretion in this species.

Qualitative changes in proteins contained in outer membrane vesicles produced by Rhizobium etli grown in the presence of the nod gene inducer naringenin.

In this work, we compared the proteomic profiles of outer membrane vesicles (OMVs) isolated from Rhizobium etli CE3 grown in minimal medium (MM) with and without exogenous naringenin. One-hundred and seven proteins were present only in OMVs from naringenin-containing cultures (N-OMVs), 57 proteins were unique to OMVs from control cultures lacking naringenin (C-OMVs) and 303 proteins were present in OMVs from both culture conditions (S-OMVs). Although we found no absolute predominance of specific types of proteins in the N-, C- or S-OMV classes, there were categories of proteins that were significantly less or more common in the different OMV categories. Proteins for energy production, translation and membrane and cell wall biogenesis were overrepresented in C-OMVs relative to N-OMVs. Proteins for carbohydrate metabolism and transport and those classified as either general function prediction only, function unknown, or without functional prediction were more common in N-OMVs than C-OMVs. This indicates that naringenin increased the proportion of these proteins in the OMVs, although NodD binding sites were only slightly more common in the promoters of genes for proteins found in the N-OMVs. In addition, OMVs from naringenin-containing cultures contained nodulation factor.

Polyamines are required for normal growth in Sinorhizobium meliloti.

Polyamines (PAs) are ubiquitous polycations derived from basic l-amino acids whose physiological roles are still being defined. Their biosynthesis and functions in nitrogen-fixing rhizobia such as Sinorhizobium meliloti have not been extensively investigated. Thin layer chromatographic and mass spectrometric analyses showed that S. meliloti Rm8530 produces the PAs, putrescine (Put), spermidine (Spd) and homospermidine (HSpd), in their free forms and norspermidine (NSpd) in a form bound to macromolecules. The S. meliloti genome encodes two putative ornithine decarboxylases (ODC) for Put synthesis. Activity assays with the purified enzymes showed that ODC2 (SMc02983) decarboxylates both ornithine and lysine. ODC1 (SMa0680) decarboxylates only ornithine. An odc1 mutant was similar to the wild-type in ODC activity, PA production and growth. In comparison to the wild-type, an odc2 mutant had 45 % as much ODC activity and its growth rates were reduced by 42, 14 and 44 % under non-stress, salt stress or acid stress conditions, respectively. The odc2 mutant produced only trace levels of Put, Spd and HSpd. Wild-type phenotypes were restored when the mutant was grown in cultures supplemented with 1 mM Put or Spd or when the odc2 gene was introduced in trans. odc2 gene expression was increased under acid stress and reduced under salt stress and with exogenous Put or Spd. An odc1 odc2 double mutant had phenotypes similar to the odc2 mutant. These results indicate that ODC2 is the major enzyme for Put synthesis in S. meliloti and that PAs are required for normal growth in vitro.

Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water.

Four new X-ray structures of tryptophan synthase (TS) crystallized with varying numbers of the amphipathic N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) molecule are presented. These structures show one of the F6 ligands threaded into the tunnel from the ß-site and reveal a distinct hydrophobic region. Over this expanse, the interactions between F6 and the tunnel are primarily nonpolar, while the F6 phosphoryl group fits into a polar pocket of the ß-subunit active site. Further examination of TS structures reveals that one portion of the tunnel (T1) binds clusters of water molecules, whereas waters are not observed in the nonpolar F6 binding region of the tunnel (T2). MD simulation of another TS structure with an unobstructed tunnel also indicates the T2 region of the tunnel excludes water, consistent with a dewetted state that presents a significant barrier to the transfer of water into the closed ß-site. We conclude that hydrophobic molecules can freely diffuse between the α- and ß-sites via the tunnel, while water does not. We propose that exclusion of water serves to inhibit reaction of water with the α-aminoacrylate intermediate to form ammonium ion and pyruvate, a deleterious side reaction in the αß-catalytic cycle. Finally, while most TS structures show ßPhe280 partially blocking the tunnel between the α- and ß-sites, new structures show an open tunnel, suggesting the flexibility of the ßPhe280 side chain. Flexible docking studies and MD simulations confirm that the dynamic behavior of ßPhe280 allows unhindered transfer of indole through the tunnel, therefore excluding a gating role for this residue.

The naringenin-induced exoproteome of Rhizobium etli CE3.

Flavonoids excreted by legume roots induce the expression of symbiotically essential nodulation (nod) genes in rhizobia, as well as that of specific protein export systems. In the bean microsymbiont Rhizobium etli CE3, nod genes are induced by the flavonoid naringenin. In this study, we identified 693 proteins in the exoproteome of strain CE3 grown in minimal medium with or without naringenin, with 101 and 100 exoproteins being exclusive to these conditions, respectively. Four hundred ninety-two (71%) of the extracellular proteins were found in both cultures. Of the total exoproteins identified, nearly 35% were also present in the intracellular proteome of R. etli bacteroids, 27% had N-terminal signal sequences and a significant number had previously demonstrated or possible novel roles in symbiosis, including bacterial cell surface modification, adhesins, proteins classified as MAMPs (microbe-associated molecular patterns), such as flagellin and EF-Tu, and several normally cytoplasmic proteins as Ndk and glycolytic enzymes, which are known to have extracellular "moonlighting" roles in bacteria that interact with eukaryotic cells. It is noteworthy that the transmembrane ß (1,2) glucan biosynthesis protein NdvB, an essential symbiotic protein in rhizobia, was found in the R. etli naringenin-induced exoproteome. In addition, potential binding sites for two nod-gene transcriptional regulators (NodD) occurred somewhat more frequently in the promoters of genes encoding naringenin-induced exoproteins in comparison to those ofexoproteins found in the control condition.

Catalytic roles of ßLys87 in tryptophan synthase: (15)N solid state NMR studies.

The proposed mechanism for tryptophan synthase shows ßLys87 playing multiple catalytic roles: it bonds to the PLP cofactor, activates C4' for nucleophilic attack via a protonated Schiff base nitrogen, and abstracts and returns protons to PLP-bound substrates (i.e. acid-base catalysis). ε-¹5N-lysine TS was prepared to access the protonation state of ßLys87 using ¹5N solid-state nuclear magnetic resonance (SSNMR) spectroscopy for three quasi-stable intermediates along the reaction pathway. These experiments establish that the protonation state of the ε-amino group switches between protonated and neutral states as the ß-site undergoes conversion from one intermediate to the next during catalysis, corresponding to mechanistic steps where this lysine residue has been anticipated to play alternating acid and base catalytic roles that help steer reaction specificity in tryptophan synthase catalysis. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. Guest Editors: Andrea Mozzarelli and Loredano Pollegioni.

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity.

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the ß-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4' of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for ß-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.

Antifungal performance of extracellular chitinases and culture supernatants of Streptomyces galilaeus CFFSUR-B12 against Mycosphaerella fijiensis Morelet.

The tropical and mycoparasite strain Streptomyces galilaeus CFFSUR-B12 was evaluated as an antagonist of Mycosphaerella fijiensis Morelet, causal agent of the Black Sigatoka Disease (BSD) of banana. On zymograms of CFFSUR-B12 culture supernatants, we detected four chitinases of approximately 32 kDa (Chi32), 20 kDa (Chi20), and two with masses well over 170 kDa (ChiU) that showed little migration during denaturing electrophoresis at different concentrations of polyacrylamide. The thymol-sulphuric acid assay showed that the ChiU were glycosylated chitinases. Moreover, matrix assisted laser desorption ionization time-of-flight MS analysis revealed that the ChiU are the same protein and identical to a family 18 chitinase from Streptomyces sp. S4 (gi|498328075). Chi32 was similar to an extracellular protein from Streptomyces albus J1074 (gi|478687481) and Chi20 was non-significantly similar to chitinases from five different strains of Streptomyces (P > 0.05). Subsequently, Chi32 and Chi20 were partially purified by anion exchange and hydrophobic interaction chromatography and tested against M. fijiensis. Chitinases failed to inhibit ascospore germination, but inhibited up to 35 and 62% of germ tube elongation and mycelial growth, respectively. We found that crude culture supernatant and living cells of S. galilaeus CFFSUR-B12 were the most effective in inhibiting M. fijiensis and are potential biocontrol agents of BSD.

Solution-State (17)O†Quadrupole Central-Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase.

Oxygen is an essential participant in the acid-base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution-state (17)O quadrupole central-transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase, reactions of the α-aminoacrylate intermediate with the nucleophiles indoline and 2-aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate-cofactor π-bonding network, particularly at the adjacent α-carbon site.

Genetic and biochemical characterization of arginine biosynthesis in Sinorhizobium meliloti 1021.

L-Ornithine production in the alfalfa microsymbiont Sinorhizobium meliloti occurs as an intermediate step in arginine biosynthesis. Ornithine is required for effective symbiosis but its synthesis in S. meliloti has been little studied. Unlike most bacteria, S. meliloti 1021 is annotated as encoding two enzymes producing ornithine: N-acetylornithine (NAO) deacetylase (ArgE) hydrolyses NAO to acetate and ornithine, and glutamate N-acetyltransferase (ArgJ) transacetylates l-glutamate with the acetyl group from NAO, forming ornithine and N-acetylglutamate (NAG). NAG is the substrate for the second step of arginine biosynthesis catalysed by NAG kinase (ArgB). Inactivation of argB in strain 1021 resulted in arginine auxotrophy. The activity of purified ArgB was significantly inhibited by arginine but not by ornithine. The purified ArgJ was highly active in NAO deacetylation/glutamate transacetylation and was significantly inhibited by ornithine but not by arginine. The purified ArgE protein (with a 6His-Sumo affinity tag) was also active in deacetylating NAO. argE and argJ single mutants, and an argEJ double mutant, are arginine prototrophs. Extracts of the double mutant contained aminoacylase (Ama) activity that deacetylated NAO to form ornithine. The purified products of three candidate ama genes (smc00682 (hipO1), smc02256 (hipO2) and smb21279) all possessed NAO deacetylase activity. hipO1 and hipO2, but not smb21279, expressed in trans functionally complemented an Escherichia coli ΔargE : : Km mutant. We conclude that Ama activity accounts for the arginine prototrophy of the argEJ mutant. Transcriptional assays of argB, argE and argJ, fused to a promoterless gusA gene, showed that their expression was not significantly affected by exogenous arginine or ornithine.

Protonation states of the tryptophan synthase internal aldimine active site from solid-state NMR spectroscopy: direct observation of the protonated Schiff base linkage to pyridoxal-5'-phosphate.

The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional (13)C, (15)N, and (31)P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.

NMR crystallography of enzyme active sites: probing chemically detailed, three-dimensional structure in tryptophan synthase.

NMR crystallography--the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry--offers unprecedented insight into three-dimensional, chemically detailed structure. Initially, researchers used NMR crystallography to refine diffraction data from organic and inorganic solids. Now we are applying this technique to explore active sites in biomolecules, where it reveals chemically rich detail concerning the interactions between enzyme site residues and the reacting substrate. Researchers cannot achieve this level of detail from X-ray, NMR,or computational methodologies in isolation. For example, typical X-ray crystal structures (1.5-2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate but do not directly identify the protonation states. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but they rely on researcher-specified chemical details. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which scientists can develop models of the active site using computational chemistry; they can then distinguish these models by comparing calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at the highest possible resolution. In this Account, we detail our first steps in the development of NMR crystallography applied to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the ß-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation.

Potentiation of the synergistic activities of chitinases ChiA, ChiB and ChiC from Serratia marcescens CFFSUR-B2 by chitobiase (Chb) and chitin binding protein (CBP).

With the goal of understanding the chitinolytic mechanism of the potential biological control strain Serratia marcescens CFFSUR-B2, genes encoding chitinases ChiA, ChiB and ChiC, chitobiase (Chb) and chitin binding protein (CBP) were cloned, the protein products overexpressed in Escherichia coli as 6His-Sumo fusion proteins and purified by affinity chromatography. Following affinity tag removal, the chitinolytic activity of the recombinant proteins was evaluated individually and in combination using colloidal chitin as substrate. ChiB and ChiC were highly active while ChiA was inactive. Reactions containing both ChiB and ChiC showed significantly increased N-acetylglucosamine trimer and dimer formation, but decreased monomer formation, compared to reactions with either enzyme alone. This suggests that while both ChiB and ChiC have a general affinity for the same substrate, they attack different sites and together degrade chitin more efficiently than either enzyme separately. Chb and CBP in combination with ChiB and ChiC (individually or together) increased their chitinase activity. We report for the first time the potentiating effect of Chb on the activity of the chitinases and the synergistic activity of a mixture of all five proteins (the three chitinases, Chb and CBP). These results contribute to our understanding of the mechanism of action of the chitinases produced by strain CFFSUR-B2 and provide a molecular basis for its high potential as a biocontrol agent against fungal pathogens.