Benzo[d]thiazole-2-carboxamides as new antituberculosis chemotypes inhibiting mycobacterial ATP phosphoribosyl transferase.

Aims: The screening of antimycobacterial benzo[d]thiazole-2-carboxamides against ATP-phosphoribosyl transferase (ATP-PRTase) was conducted. Materials & methods: The antitubercular potential of compounds 1 and 2 against ATP-PRTase was assessed through the determination of half maximal effective concentration (EC50) and binding constant (Kd), as well as competitive inhibitory studies and studies of perturbation of secondary structure, molecular modeling and L-histidine complementation assay. Results & conclusion: Compounds 1n and 2a significantly inhibited ATP-PRTase as evidenced by their EC50 and Kd values and the perturbation of the secondary structure study. Compound 1n exhibited stronger competitive inhibition toward ATP compared with 2a. The inhibition of the growth of Mycobacterium tuberculosis by targeting the L-histidine biosynthesis pathway and molecular modeling studies further supported the inhibition of ATP-PRTase.

Allosteric Inhibition of Acinetobacter baumannii ATP Phosphoribosyltransferase by Protein:Dipeptide and Protein:Protein Interactions.

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis in bacteria, namely, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-ß-d-ribosyl)-ATP (PRATP) and pyrophosphate. Catalytic (HisGS) and regulatory (HisZ) subunits assemble in a hetero-octamer where HisZ activates HisGS and mediates allosteric inhibition by histidine. In Acinetobacter baumannnii, HisGS is necessary for the bacterium to persist in the lung during pneumonia. Inhibition of ATPPRT is thus a promising strategy for specific antibiotic development. Here, A. baumannii ATPPRT is shown to follow a rapid equilibrium random kinetic mechanism, unlike any other ATPPRT. Histidine noncompetitively inhibits ATPPRT. Binding kinetics indicates histidine binds to free ATPPRT and to ATPPRT:PRPP and ATPPRT:ATP binary complexes with similar affinity following a two-step binding mechanism, but with distinct kinetic partition of the initial enzyme:inhibitor complex. The dipeptide histidine-proline inhibits ATPPRT competitively and likely uncompetitively, respectively, against PRPP and ATP. Rapid kinetics analysis shows His-Pro binds to the ATPPRT:ATP complex via a two-step binding mechanism. A related HisZ that shares 43% sequence identity with A. baumannii HisZ is a tight-binding allosteric inhibitor of A. baumannii HisGS. These findings lay the foundation for inhibitor design against A. baumannii ATPPRT.

Mapping the Structural Path for Allosteric Inhibition of a Short-Form ATP Phosphoribosyltransferase by Histidine.

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis, being allosterically inhibited by the final product of the pathway. Allosteric inhibition of long-form ATPPRTs by histidine has been extensively studied, but inhibition of short-form ATPPRTs is poorly understood. Short-form ATPPRTs are hetero-octamers formed by four catalytic subunits (HisGS) and four regulatory subunits (HisZ). HisGS alone is catalytically active and insensitive to histidine. HisZ enhances catalysis by HisGS in the absence of histidine but mediates allosteric inhibition in its presence. Here, steady-state and pre-steady-state kinetics establish that histidine is a noncompetitive inhibitor of short-form ATPPRT and that inhibition does not occur by dissociating HisGS from the hetero-octamer. The crystal structure of ATPPRT in complex with histidine and the substrate 5-phospho-α-d-ribosyl-1-pyrophosphate was determined, showing histidine bound solely to HisZ, with four histidine molecules per hetero-octamer. Histidine binding involves the repositioning of two HisZ loops. The histidine-binding loop moves closer to histidine to establish polar contacts. This leads to a hydrogen bond between its Tyr263 and His104 in the Asp101-Leu117 loop. The Asp101-Leu117 loop leads to the HisZ-HisGS interface, and in the absence of histidine, its motion prompts HisGS conformational changes responsible for catalytic activation. Following histidine binding, interaction with the histidine-binding loop may prevent the Asp101-Leu117 loop from efficiently sampling conformations conducive to catalytic activation. Tyr263Phe-PaHisZ-containing PaATPPRT, however, is less susceptible though not insensitive to histidine inhibition, suggesting the Tyr263-His104 interaction may be relevant to yet not solely responsible for transmission of the allosteric signal.

Hinge Twists and Population Shifts Deliver Regulated Catalysis for ATP-PRT in Histidine Biosynthesis.

Allosteric regulation plays an important role in the control of metabolic flux in biosynthetic pathways. In microorganisms, many enzymes in these pathways adopt different strategies of allostery to allow the tuning of their activities in response to metabolic demand. Thus, it is important to uncover the mechanism of allosteric signal transmission to fully comprehend the complex control of enzyme function and its evolution. ATP-phosphoribosyltransferase (ATP-PRT), as the first enzyme in the histidine biosynthetic pathway, is allosterically regulated by histidine and offers a good platform for the study of allostery. Two forms of ATP-PRT, namely long and short forms, were discovered that show different arrangements of their regulatory machinery. Crystal structures of the long-form ATP-PRT have revealed overall conformational changes in the inhibited state, but the observed changes in the active state are quite subtle, making the elucidation of its allosteric mechanism difficult. Here, we combine computational methods (ligand docking, quantum mechanics/molecular mechanics optimization, and molecular dynamic simulations) with experimental studies to probe the signal transmission between remote allosteric and active sites. Our results reveal that distinct conformational ensembles of the catalytic domain with different dynamic properties exist in the ligand-free and histidine-bound enzymes. These ensembles display different capabilities in supporting the catalytic and allosteric function of ATP-PRT. The findings give insight into the underlying mechanism of allostery and allow us to propose that the hinge twisting within the catalytic domain is the key for both enhancement of catalysis and provision of regulation in ATP-PRT enzymes.

Guarding the gateway to histidine biosynthesis in plants: Medicago truncatula ATP-phosphoribosyltransferase in relaxed and tense states.

In the first committed step of histidine biosynthesis, adenosine 5'-triphosphate (ATP) and 5-phosphoribosyl-α1-pyrophosphate (PRPP), in the presence of ATP phosphoribosyltransferase (ATP-PRT, EC 2.4.2.17), yield phosphoribosyl-ATP. ATP-PRTs are subject to feedback inhibition by histidine that allosterically binds between the regulatory domains. Histidine biosynthetic pathways of bacteria, lower eukaryotes, and plants are considered promising targets for the design of antibiotics, antifungal agents, and herbicides because higher organisms are histidine heterotrophs. Plant ATP-PRTs are similar to one of the two types of their bacterial counterparts, the long-type ATP-PRTs. A biochemical and structural study of ATP-PRT from the model legume plant, Medicago truncatula (MedtrATP-PRT1) is reported herein. Two crystal structures, presenting homohexameric MedtrATP-PRT1 in its relaxed (R-) and histidine-bound, tense (T-) states allowed to observe key features of the enzyme and provided the first structural insights into an ATP-PRT from a eukaryotic organism. In particular, they show pronounced conformational reorganizations during R-state to T-state transition that involves substantial movements of domains. This rearrangement requires a trans- to cis- switch of a peptide backbone within the hinge region of MedtrATP-PRT1. A C-terminal α-helix, absent in bacteria, reinforces the hinge that is constituted by two peptide strands. As a result, conformations of the R- and T-states are significantly different from the corresponding states of prokaryotic enzymes with known 3-D structures. Finally, adenosine 5'-monophosphate (AMP) bound at the active site is consistent with a competitive (and synergistic with histidine) nature of AMP inhibition.

Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase.

Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg2+-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-ß-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisGS is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS's catalytic rate, but decreases the hetero-octamer's, indicating that chemistry and product release are rate-limiting for HisGS and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.

A dimeric catalytic core relates the short and long forms of ATP-phosphoribosyltransferase.

Adenosine triphosphate (ATP) phosphoribosyltransferase (ATP-PRT) catalyses the first committed step of histidine biosynthesis in plants and microorganisms. Two forms of ATP-PRT have been reported, which differ in their molecular architecture and mechanism of allosteric regulation. The short-form ATP-PRT is a hetero-octamer, with four HisG chains that comprise only the catalytic domains and four separate chains of HisZ required for allosteric regulation by histidine. The long-form ATP-PRT is homo-hexameric, with each chain comprising two catalytic domains and a covalently linked regulatory domain that binds histidine as an allosteric inhibitor. Here, we describe a truncated long-form ATP-PRT from Campylobacter jejuni devoid of its regulatory domain (CjeATP-PRTcore). Results showed that CjeATP-PRTcore is dimeric, exhibits attenuated catalytic activity, and is insensitive to histidine, indicating that the covalently linked regulatory domain plays a role in both catalysis and regulation. Crystal structures were obtained for CjeATP-PRTcore in complex with both substrates, and for the first time, the complete product of the reaction. These structures reveal the key features of the active site and provide insights into how substrates move into position during catalysis.

Transition State Analysis of Adenosine Triphosphate Phosphoribosyltransferase.

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first step in histidine biosynthesis, a pathway essential to microorganisms and a validated target for antimicrobial drug design. The ATP-PRT enzyme catalyzes the reversible substitution reaction between phosphoribosyl pyrophosphate and ATP. The enzyme exists in two structurally distinct forms, a short- and a long-form enzyme. These forms share a catalytic core dimer but bear completely different allosteric domains and thus distinct quaternary assemblies. Understanding enzymatic transition states can provide essential information on the reaction mechanisms and insight into how differences in domain structure influence the reaction chemistry, as well as providing a template for inhibitor design. In this study, the transition state structures for ATP-PRT enzymes from Campylobacter jejuni and Mycobacterium tuberculosis (long-form enzymes) and from Lactococcus lactis (short-form) were determined and compared. Intrinsic kinetic isotope effects (KIEs) were obtained at reaction sensitive positions for the reverse reaction using phosphonoacetic acid, an alternative substrate to the natural substrate pyrophosphate. The experimental KIEs demonstrated mechanistic similarities between the three enzymes and provided experimental boundaries for quantum chemical calculations to characterize the transition states. Predicted transition state structures support a dissociative reaction mechanism with a DN*AN‡ transition state. Weak interactions from the incoming nucleophile and a fully dissociated ATP adenine are predicted regardless of the difference in overall structure and quaternary assembly. These studies establish that despite significant differences in the quaternary assembly and regulatory machinery between ATP-PRT enzymes from different sources, the reaction chemistry and catalytic mechanism are conserved.

Uncoupling conformational states from activity in an allosteric enzyme.

ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-L-alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and L-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited L-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.Active and inactive state ATP-phosphoribosyltransferases (ATP-PRTs) are believed to have different conformations. Here the authors show that in both states, ATP-PRT has a similar structural arrangement, suggesting that dynamic alterations are involved in ATP-PRT regulation by allosteric modulators.

Hybrid Mass Spectrometry Approaches to Determine How L-Histidine Feedback Regulates the Enzyzme MtATP-Phosphoribosyltransferase.

MtATP-phosphoribosyltransferase (MtATP-PRT) is an enzyme catalyzing the first step of the biosynthesis of L-histidine in Mycobacterium tuberculosis, and proposed to be regulated via an allosteric mechanism. Native mass spectrometry (MS) reveals MtATP-PRT to exist as a hexamer. Conformational changes induced by L-histidine binding and the influence of buffer pH are determined with ion mobility MS, hydrogen deuterium exchange (HDX) MS, and analytical ultracentrifugation. The experimental collision cross-section (DTCCSHe) decreases from 76.6 to 73.5 nm2 upon ligand binding at pH 6.8, which correlates to the decrease in CCS calculated from crystal structures. No such changes in conformation were found at pH 9.0. Further detail on the regions that exhibit conformational change on L-histidine binding is obtained with HDX-MS experiments. On incubation with L-histidine, rapid changes are observed within domain III, and around the active site at longer times, indicating an allosteric effect.

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase.

Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyzes the first step in histidine biosynthesis, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to generate N1-(5-phospho-ß-d-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was determined at 2.34 Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both the ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-4-fold increase in kcat. Reaction profiles for both enzymes cannot be distinguished by 31P nuclear magnetic resonance, indicating that the same reaction is catalyzed. The temperature dependence of kcat shows deviation from Arrhenius behavior at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterization of a psychrophilic ATPPRT.

Independent catalysis of the short form HisG from Lactococcus lactis.

ATP-phosphoribosyltransferase (ATP-PRT) catalyses the first step of histidine biosynthesis. Two different forms of ATP-PRT have been described; the homo-hexameric long form, and the hetero-octameric short form. Lactococcus lactis possesses the short form ATP-PRT comprising four subunits of HisGS , the catalytic subunit, and four subunits of HisZ, a histidyl-tRNA synthetase paralogue. Previous studies have suggested that HisGS requires HisZ for catalysis. Here, we reveal that the dimeric HisGS does display ATP-PRT activity in the absence of HisZ. This result reflects the evolutionary relationship between the long and short form ATP-PRT, which acquired allosteric inhibition and enhanced catalysis via two divergent strategies.

ATP phosphoribosyltransferase from symbiont Entomomyces delphacidicola invovled in histidine biosynthesis of Nilaparvata lugens (Stål).

Histidine is an essential amino acid assumed to be synthesized by an obligatory yeast-like symbiont (Entomomyces delphacidicola str. NLU) in Nilaparvata lugens, an important rice pest. The adenosine-triphosphate phosphoribosyltransferase (ATP-PRTase) facilities the committed first step of the histidine biosynthesis pathway. In the current study, a putative ATP-PRTase was cloned and verified to be of E. delphacidicola origin (EdePRTase). The expression of the gene was spatial and temporal universal with a profile that matched the distribution of the fungal symbiont. RNA interference aided the knockdown of the EdePRTase-suppressed EdePRTase expression by 32-48 %. Hemolymph histidine level was also reduced followed by significant reduction of adult body weight. However, other performance characters including nymph development, survival, and adult sex ratio were not adversely affected by the knockdown. Furthermore, forced histidine exposure (through injection or feeding) significantly inhibited the EdePRTase mRNA levels at higher concentrations, but significantly increased EdePRTase expression levels at lower concentrations (feeding only). The significance of these findings support that the EdePRTase is from symbiont E. delphacidicola, and its involvement in histidine biosynthesis of N. lugens was discussed. The results provide a better understanding of EdePRTase and the encoded functional ATP-PRTase enzyme regulation in N. lugens and insects in general.

Campylobacter jejuni adenosine triphosphate phosphoribosyltransferase is an active hexamer that is allosterically controlled by the twisting of a regulatory tail.

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisGL ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme.

Corynebacterium glutamicum ATP-phosphoribosyl transferases suitable for L-histidine production--Strategies for the elimination of feedback inhibition.

L-Histidine biosynthesis in Corynebacterium glutamicum is mainly regulated by L-histidine feedback inhibition of the ATP-phosphoribosyltransferase HisG that catalyzes the first step of the pathway. The elimination of this feedback inhibition is the first and most important step in the development of an L-histidine production strain. For this purpose, a combined approach of random mutagenesis and rational enzyme redesign was performed. Mutants spontaneously resistant to the toxic L-histidine analog ß-(2-thiazolyl)-DL-alanine (2-TA) revealed novel and unpredicted mutations in the C-terminal regulatory domain of HisG resulting in increased feedback resistance. Moreover, deletion of the entire C-terminal regulatory domain in combination with the gain of function mutation S143F in the catalytic domain resulted in a HisG variant that is still highly active even at L-histidine concentrations close to the solubility limit. Notably, the S143F mutation on its own provokes feedback deregulation, revealing for the first time an amino acid residue in the catalytic domain of HisG that is involved in the feedback regulatory mechanism. In addition, we investigated the effect of hisG mutations for L-histidine production on different levels. This comprised the analysis of different expression systems, including plasmid- and chromosome-based overexpression, as well as the importance of codon choice for HisG mutations. The combination of domain deletions, single amino acid exchanges, codon choice, and chromosome-based overexpression resulted in production strains accumulating around 0.5 g l(-1) L-histidine, demonstrating the added value of the different approaches.

Taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways.

Enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. Here we present an approach to rapidly generate sets of enzymes overriding this control. It is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. The sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened the library in a strain carrying the sensor pSenLys-Spc, which detects l-lysine, l-arginine and l-histidine. Six of the resulting N-acetyl-l-glutamate kinase proteins were further developed and characterized and found to be at least 20-fold less sensitive toward l-arginine inhibition than the wild-type enzyme. Overexpression of the mutein ArgB-K47H-V65A in C. glutamicumΔargR led to the accumulation of 34 mM l-arginine in the culture medium. We also screened mutant libraries of lysC-encoded aspartate kinase and hisG-encoded ATP phosphoribosyltransferase. We isolated 11 LysC muteins, enabling up to 45 mM l-lysine accumulation, and 13 HisG muteins, enabling up to 17 mM l-histidine accumulation. These results demonstrate that in vivo screening of enzyme libraries by using metabolite sensors is extremely well suited to identify high-performance muteins required for overproduction.

Targeting the histidine pathway in Mycobacterium tuberculosis.

Worldwide, tuberculosis is the leading cause of morbidity and mortality due to a single bacterial pathogen, Mycobacterium tuberculosis (Mtb). The increasing prevalence of this disease, the emergence of multi-, extensively, and totally drug-resistant strains, complicated by co-infection with the human immunodeficiency virus, and the length of tuberculosis chemotherapy have led to an urgent and continued need for the development of new and more effective antitubercular drugs. Within this context, the L-histidine biosynthetic pathway, which converts 5-phosphoribosyl 1-pyrophosphate to L-histidine in ten enzymatic steps, has been reported as a promising target of antimicrobial agents. This pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants but is absent in mammals, making these enzymes highly attractive targets for the drug design of new antimycobacterial compounds with selective toxicity. Moreover, the biosynthesis of L-histidine has been described as essential for Mtb growth in vitro. Accordingly, a comprehensive overview of Mycobacterium tuberculosis histidine pathway enzymes as attractive targets for the development of new antimycobacterial agents is provided, mainly summarizing the previously reported inhibition data for Mtb or orthologous proteins.

[The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants].

Strain MG 1655+hisGr hisL'-Delta, purR, which produces histidine with a weight yield of approximately 12% from glucose, was constructed through directed chromosomal modifications of the laboratory Escherichia coli strain MG 1655+, which has a known genome sequence. A feedback-resistant ATP-phosphoribosyl transferase encoded by the mutant hisGr (E271 K) was the main determinant of histidine production. A further increase in histidine production was achieved by the expression enhance of a mutant his operon containing hisGr through the deleting attenuator region (hisL'-Delta). An increase in the expression of the wildtype his operon did not result in histidine accumulation. Deletion of the transcriptional regulator gene purR increased the biomass produced and maintained the level of histidine production per cell under the fermentation conditions used.

Modification of histidine biosynthesis pathway genes and the impact on production of L-histidine in Corynebacterium glutamicum.

Histidine biosynthesis in Corynebacterium glutamicum is regulated not only by feedback inhibition by the first enzyme in the pathway, but also by repression control of the synthesis of the histidine enzymes. C. glutamicum histidine genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. We constructed plasmid pK18hisDPtac to replace the native hisD promoter with the tac promoter, and overexpressed phosphoribosyl-ATP-pyrophosphohydrolase, encoded by hisE, and ATP-phosphoribosyltransferase, encoded by hisG. The L-histidine titer at 0.85 g l(-1) was 80 % greater in the transformed bacterium and production of byproducts, L-alanine and L-tryptophan, was significantly decreased. However, accumulation of glutamic acid increased by 58 % (2.8 g l(-1)). This study represents the first attempt to substitute the histidine biosynthesis pathway promoter in the chromosome with a stronger promoter to increase histidine production.

Mechanism of feedback allosteric inhibition of ATP phosphoribosyltransferase.

MtATP-phosphoribosyltransferase catalyzes the first and committed step in l-histidine biosynthesis in Mycobacterium tuberculosis and is therefore subjected to allosteric feedback regulation. Because of its essentiality, this enzyme is being studied as a potential target for novel anti-infectives. To understand the basis for its regulation, we characterized the allosteric inhibition using gel filtration, steady-state and pre-steady-state kinetics, and the pH dependence of inhibition and binding. Gel filtration experiments indicate that MtATP-phosphoribosyltransferase is a hexamer in solution, in the presence or absence of l-histidine. Steady-state kinetic studies demonstrate that l-histidine inhibition is uncompetitive versus ATP and noncompetitive versus PRPP. At pH values close to neutrality, a K(ii) value of 4 µM was obtained for l-histidine. Pre-steady-state kinetic experiments indicate that chemistry is not rate-limiting for the overall reaction and that l-histidine inhibition is caused by trapping the enzyme in an inactive conformation. The pH dependence of binding, obtained by nuclear magnetic resonance, indicates that l-histidine binds better as the neutral α-amino group. The pH dependence of inhibition (K(ii)), on the contrary, indicates that l-histidine better inhibits MtATP-phosphoribosytransferase with a neutral imidazole and an ionized α-amino group. These results are combined into a model that accounts for the allosteric inhibition of MtATP-phosphoribosyltransferase.