Chemoenzymatic Synthesis of Select Intermediates and Natural Products of the Desferrioxamine E Siderophore Pathway.

The NIS synthetase family of enzymes responsible for the biosynthesis of siderophores is increasingly associated with bacterial virulence. Proteins in this class represent outstanding potential drug targets, assuming that basic biochemical and structural characterizations can be completed. Towards this goal, we have mated an improved synthesis of the non-commercial amino acid N-hydroxy-N-succinylcadaverine (HSC, 6) with an isothermal titration calorimetry (ITC) assay that profiles the iterative stages of HSC trimerization and macrocyclization by NIS synthetase DesD from Streptomyces coelicolor. HSC synthesis begins with multigram-scale Gabrielle and tert-butyl N-(benzyloxy)carbamate alkylations of 1-bromo-5-chloropentane following prior literature, but the end-game reported herein has two advantages for greater material throughput: (1) hydrogenolysis of benzyl ether and Cbz blocking groups is best accomplished with Pearlman's catalyst at 40 psi of H2 and (2) purification of neutral (zwitterionic) HSC is effected by simple flash chromatography over silica gel in MeOH. HSC is subsequently shown to be a substrate for NIS synthetase DesD, which catalyzes three successive amide bond syntheses via adenyl monophosphate ester intermediates. We quantify and present the iterative and overall enzyme kinetic constants associated with formation of the cyclotrimeric siderophore desferrioxamine E (dfoE, 1).

A procedure for removal of cyanuric acid in swimming pools using a cell-free thermostable cyanuric acid hydrolase.

Cyanuric acid (CYA) is used commercially for maintaining active chlorine to inactivate microbial and viral pathogens in swimming pools and hot tubs. Repeated CYA addition can cause a lack of available chlorine and adequate disinfection. Acceptable CYA levels can potentially be restored via cyanuric acid hydrolases (CAH), enzymes that hydrolyze CYA to biuret under mild conditions. Here we describe a previously unknown CAH enzyme from Pseudolabrys sp. Root1462 (CAH-PR), mined from public databases by bioinformatic analysis of potential CAH genes, which we show to be suitable in a cell-free form for industrial applications based upon favorable enzymatic and physical properties, combined with high-yield expression in aerobic cell culture. The kinetic parameters and modeled structure were similar to known CAH enzymes, but the new enzyme displayed a surprising thermal and storage stability. The new CAH enzyme was applied, following addition of inexpensive sodium sulfite, to hydrolyze CYA to biuret. At the desired endpoint, hypochlorite addition inactivated remaining enzyme and oxidized biuret to primarily dinitrogen and carbon dioxide gases. The mechanism of biuret oxidation with hypochlorite under conditions relevant to recreational pools is described.

Cofactor Complexes of DesD, a Model Enzyme in the Virulence-related NIS Synthetase Family.

The understudied nonribosomal-peptide-synthetase-independent siderophore (NIS) synthetase family has been increasingly associated with virulence in bacterial species due to its key role in the synthesis of hydroxamate and carboxylate "stealth" siderophores. We have identified a model family member, DesD, from Streptomyces coelicolor, to structurally characterize using a combination of a wild-type and a Arg306Gln variant in apo, cofactor product AMP-bound, and cofactor reactant ATP-bound complexes. The kinetics in the family has been limited by solubility and reporter assays, so we have developed a label-free kinetics assay utilizing a single-injection isothermal-titration-calorimetry-based method. We report second-order rate constants that are 50 times higher than the previous estimations for DesD. Our Arg306Gln DesD variant was also tested under identical buffer and substrate conditions, and its undetectable activity was confirmed. These are the first reported structures for DesD, and they describe the critical cofactor coordination. This is also the first label-free assay to unambiguously determine the kinetics for an NIS synthetase.

Allosteric reversion of Haemophilus influenzae ß-carbonic anhydrase via a proline shift.

Haemophilus influenzae ß-carbonic anhydrase (HICA) has been reverse-engineered in the allosteric site region to resemble the nonallosteric Pisum sativum enzyme in order to identify critical features of allostery and intersusbunit communication. Three variants (W39V/G41A, P48S/A49P, and W39V/G41A/P48S/A49P) were identified, through a comparison with a crystal structure of nonallosteric P. sativum ß-carbonic anhydrase (PSCA, PDB 1EKJ ), to potentially revert HICA to a nonallosteric enzyme. The W39V/G41A and P48S/A49P mutations decreased the apparent kcat/Km proton dependence from 4 to 2 and 1, respectively, increasing the overall maximal kcat/Km to 16 ± 2 µM(-1) s(-1) (380% of wild type) and 17 ± 3 µM(-1) s(-1) (405% of wild type). The pKa values of the metal-bound water molecule based on the pH-rate profile kinetics (8.32 ± 0.04 for W39V/G41A and 8.3 ± 0.1 for P48S/A49P) were also slightly higher than that for the wild-type enzyme (7.74 ± 0.04). The P48S/A49P variant has lost all pH-rate cooperativity. The W39V/G41A/P48S/A49P variant's kinetics were unusual and were fit with a log-linear function with a slope 0.9 ± 0.2. The crystal structure of the W39V/G41A variant revealed an active site very similar to the T-state wild-type oligomer with bicarbonate trapped in the escort site. By contrast, the X-ray crystal structure of a proline shift variant (P48S/A49P) reveals that it has adopted an active site conformation nearly identical to that of nonallosteric ß-carbonic anhydrase (R-state) for one chain, including a tight association with the dimer-exchanged N-terminal helices; the second chain in the asymmetric unit is associated in a biologically relevant oligomer, but it adopts a T-state conformation that is not capped by dimer-exchanged N-terminal helices. The hybrid R/T nature of HICA P48S/A49P structurally recapitulates the interruption of pH-rate cooperativity observed for this variant. Comparison of the conformations of the R and T chains of P48S/A49P suggests a new hypothesis to explain HICA allosteric communication that is mediated by the N-terminal helices and anion binding at the dimer interface.

Surface histidine mutations for the metal affinity purification of a ß-carbonic anhydrase.

Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques.

Co(II)-substituted Haemophilus influenzae ß-carbonic anhydrase: spectral evidence for allosteric regulation by pH and bicarbonate ion.

Cobalt(II)-substituted Haemophilus influenzae ß-carbonic anhydrase (HICA) has been produced by overexpression in minimal media supplemented with CoCl(2), enabling kinetic, structural, and spectroscopic characterization. Co(II)-substituted HICA (Co-HICA) has comparable catalytic activity to that of wild-type enzyme with k(cat)=82±19 ms(-1) (120% of wild-type). The X-ray crystal structure of Co-HICA was determined to 2.5Å resolution, and is similar to the zinc enzyme. The absorption spectrum of Co-HICA is consistent with four-coordinate geometry. pH-dependent changes in the absorption spectrum of Co-HICA, including an increase in molar absorptivity and a red shift of a 580 nm peak with decreasing pH, correlate with the pH dependence of k(cat)/K(m). The absence of isosbestic points in the pH-dependent absorption spectra suggest that more than two absorbing species are present. The addition of bicarbonate ion at pH 8.0 triggers spectral changes in the metal coordination sphere that mimic that of lowering pH, supporting its hypothesized role as an allosteric inhibitor of HICA. Homogeneously (99±1% Co) and heterogeneously (52±5% Co) substituted Co-HICA have distinctly different colors and absorption spectra, suggesting that the metal ions in the active sites in the allosteric dimer of Co-HICA engage in intersubunit communication.

Evidence for a bicarbonate "escort" site in Haemophilus influenzae beta-carbonic anhydrase .

The Haemophilus influenzae beta-carbonic anhydrase (HICA) allosteric site variants V47A and G41A were overexpressed and purified to homogeneity. These variants have k(cat)/K(m) values similar to that of the wild-type enzyme and exhibit a similar dramatic decrease in catalytic activity at pH <8.0. However, both HICA-G41A and -V47A were serendipitously found to bind sulfate ion or bicarbonate ion near pairs of Glu50 and Arg64 residues located on the dimerization interface. In the case of HICA-V47A, bicarbonate ions simultaneously bind to both the dimerization interface and the allosteric sites. For HICA-G41A, two of 12 chains in the asymmetric unit bind bicarbonate ion exclusively at the dimerization interface, while the remaining 10 chains bind bicarbonate ion exclusively at the allosteric site. We propose that the new anion binding site along the dimerization interface of HICA is an "escort" site that represents an intermediate along the ingress and egress route of bicarbonate ion to and from the allosteric binding site, respectively. The structural evidence for sulfate binding at the escort site suggests that the mechanism of sulfate activation of HICA is the result of sulfate ion competing for bicarbonate at the escort site, preventing passage of bicarbonate from the bulk solution to its allosteric site.

Characterization of the multiple transferable resistance repressor, MtrR, from Neisseria gonorrhoeae.

MtrR represses expression of the Neisseria gonorrhoeae mtrCDE multidrug efflux transporter genes. MtrR displays salt-dependent DNA binding, a stoichiometry of two dimers per DNA site, and, for a protein that was expected to be essentially all helical, a high percentage of random coil and possibly beta-sheet structure.