Investigating the Roles of Active Site Residues in Mycobacterium tuberculosis Indole-3-glycerol Phosphate Synthase, a Potential Target for Antitubercular Agents.

Mycobacterium tuberculosis drug resistance is emerging and new drug targets are needed. Tryptophan biosynthesis is necessary for M. tuberculosis replication and virulence. Indole-3-glycerol phosphate synthase (IGPS) catalyzes a step in M. tuberculosis tryptophan biosynthesis and has been suggested as a potential anti-infective target, but our understanding of this enzyme is limited. To aid in inhibitor design and gain a greater mechanistic picture of this enzyme, there is a need to understand the roles of active site amino acids in ligand binding and catalysis. In this work, we explored the roles of conserved active site amino acids Glu57, Lys59, Lys119, Glu168, and Glu219. Mutation of each to Ala results in loss of all detectable activity. The Glu57Gln, Lys59Arg, Lys119Arg, Glu168Gln, and Glu219Asp mutations result in large activity losses, while Glu219Gln has enhanced activity. Analysis of the enzymatic data yields the following main conclusions: (A) Lys119 is the likely catalytic acid in the CdRP ring closure step. (B) Glu168 stabilizes a charged reaction intermediate and may also be the catalytic base. (C) Glu57, Glu219, and Lys119 form a closely arranged triad in which Glu57 and Glu219 modulate the pKa of Lys119, and thus overall activity. This increased understanding of inter- and intramolecular interactions and demonstration of the highly coordinated nature of the M. tuberculosis IGPS active site provide new mechanistic information and guidance for future work with this potential new drug target.

Indole-3-Glycerol Phosphate Synthase From Mycobacterium tuberculosis: A Potential New Drug Target.

Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis, affects millions of people worldwide. Several TB drugs have lost efficacy due to emerging drug resistance and new anti-TB targets are needed. Recent research suggests that indole-3-glycerol phosphate synthase (IGPS) in M. tuberculosis (MtIGPS) could be such a target. IGPS is a (ß/α)8 -barrel enzyme that catalyzes the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose 5'-phosphate (CdRP) into indole-glycerol-phosphate (IGP) in the bacterial tryptophan biosynthetic pathway. M. tuberculosis over expresses the tryptophan pathway genes during an immune response and inhibition of MtIGPS allows CD4 T-cells to more effectively fight against M. tuberculosis. Here we review the published data on MtIGPS expression, kinetics, mechanism, and inhibition. We also discuss MtIGPS crystal structures and compare them to other IGPS structures to reveal potential structure-function relationships of interest for the purposes of drug design and biocatalyst engineering.

Differences in a conformational equilibrium distinguish catalysis by the endothelial and neuronal nitric-oxide synthase flavoproteins.

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.

Reductase domain of Drosophila melanogaster nitric-oxide synthase: redox transformations, regulation, and similarity to mammalian homologues.

The nitric oxide synthase of Drosophila melanogaster (dNOS) participates in essential developmental and behavioral aspects of the fruit fly, but little is known about dNOS catalysis and regulation. To address this, we expressed a construct comprising the dNOS reductase domain and its adjacent calmodulin (CaM) binding site (dNOSr) and characterized the protein regarding its catalytic, kinetic, and regulatory properties. The Ca2+ concentration required for CaM binding to dNOSr was between that of the mammalian endothelial and neuronal NOS enzymes. CaM binding caused the cytochrome c reductase activity of dNOSr to increase 4 times and achieve an activity comparable to that of mammalian neuronal NOS. This change was associated with decreased shielding of the FMN cofactor from solvent and an increase in the rate of NADPH-dependent flavin reduction. Flavin reduction in dNOSr was relatively slow following the initial 2-electron reduction, suggesting a slow inter-flavin electron transfer, and no charge-transfer complex was observed between bound NADP+ and reduced FAD during the process. We conclude that dNOSr catalysis and regulation is most similar to the mammalian neuronal NOS reductase domain, although differences exist in their flavin reduction behaviors. The apparent conservation between the fruit fly and mammalian enzymes is consistent with dNOS operating in various signal cascades that involve NO.

Role of Asp1393 in catalysis, flavin reduction, NADP(H) binding, FAD thermodynamics, and regulation of the nNOS flavoprotein.

Nitric oxide synthases (NOS) are flavoheme enzymes with important roles in biology. The reductase domain of neuronal NOS (nNOSr) contains a widely conserved acidic residue (Asp(1393)) that is thought to facilitate hydride transfer between NADPH and FAD. Previously we found that the D1393V and D1393N mutations lowered the NO synthesis activity and the rates of heme and flavin reduction in full-length nNOS. To examine the mechanisms for these results in greater detail, we incorporated D1393V and D1393N substitutions into nNOSr along with a truncated NADPH-FAD domain construct (FNR) and characterized the mutants. D1393V nNOSr had markedly lower (

C-terminal tail residue Arg1400 enables NADPH to regulate electron transfer in neuronal nitric-oxide synthase.

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg(1400) in the C-terminal tail regulatory element and the 2'-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg(1400) in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K(m) for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg(1400)-2'-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.

The FAD-shielding residue Phe1395 regulates neuronal nitric-oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain.

Phe(1395) stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe(1395) was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP(+) binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP(+) with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP(+) dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP(+)-bound state as compared with wild type. We conclude that Phe(1395) regulates nNOSr catalysis in two ways. It facilitates NADP(+) release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer.