Structure and mechanism of PhnP, a phosphodiesterase of the carbon-phosphorus lyase pathway.

PhnP is a phosphodiesterase that plays an important role within the bacterial carbon-phosphorus lyase (CP-lyase) pathway by recycling a "dead-end" intermediate, 5-phospho-α-d-ribosyl 1,2-cyclic phosphate, that is formed during organophosphonate catabolism. As a member of the metallo-ß-lactamase superfamily, PhnP is most homologous in sequence and structure to tRNase Z phosphodiesterases. X-ray structural analysis of PhnP complexed with orthovanadate to 1.5 Å resolution revealed this inhibitor bound in a tetrahedral geometry by the two catalytic manganese ions and the putative general acid residue H200. Guided by this structure, we probed the contributions of first- and second-sphere active site residues to catalysis and metal ion binding by site-directed mutagenesis, kinetic analysis, and ICP-MS. Alteration of H200 to alanine resulted in a 6-33-fold decrease in k(cat)/K(M) with substituted methyl phenylphosphate diesters with leaving group pK(a) values ranging from 4 to 8.4. With bis(p-nitrophenyl)phosphate as a substrate, there was a 10-fold decrease in k(cat)/K(M), primarily the result of a large increase in K(M). Moreover, the nickel ion-activated H200A PhnP displayed a bell-shaped pH dependence for k(cat)/K(M) with pK(a) values (pK(a1) = 6.3; pK(a2) = 7.8) that were comparable to those of the wild-type enzyme (pK(a1) = 6.5; pK(a2) = 7.8). Such modest effects are counter to what is expected for a general acid catalyst and suggest an alternate role for H200 in this enzyme. A Brønsted analysis of the PhnP reaction with a series of substituted phenyl methyl phosphate esters yielded a linear correlation, a ß(lg) of -1.06 ± 0.1, and a Leffler α value of 0.61, consistent with a synchronous transition state for phosphoryl transfer. On the basis of these data, we propose a mechanism for PhnP.

Chloramphenicol biosynthesis: the structure of CmlS, a flavin-dependent halogenase showing a covalent flavin-aspartate bond.

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 A resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4alpha)-OOH intermediate. Strikingly, the 8alpha carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.

Structure of PhnP, a phosphodiesterase of the carbon-phosphorus lyase pathway for phosphonate degradation.

Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the beta-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(p-nitrophenyl)phosphate and 2',3'-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn(2+) and Ni(2+) for catalysis, whereas Zn(2+) afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn(2+) ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn(2+) binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.

Expression, purification and preliminary diffraction studies of CmlS.

CmlS, a flavin-dependent halogenase (FDH) present in the chloramphenicol-biosynthetic pathway in Streptomyces venezuelae, directs the dichlorination of an acetyl group. The reaction mechanism of CmlS is of considerable interest as it will help to explain how the FDH family can halogenate a wide range of substrates through a common mechanism. The protein has been recombinantly expressed in Escherichia coli and purified to homogeneity. The hanging-drop vapour-diffusion method was used to produce crystals that were suitable for X-ray diffraction. Data were collected to 2.0 A resolution. The crystal belonged to space group C2, with unit-cell parameters a = 208.1, b = 57.7, c = 59.9 A, beta = 97.5 degrees .

Expression, purification and preliminary diffraction studies of PhnP.

PhnP belongs to a 14-gene operon that supports the growth of Escherichia coli on alkylphosphonates as a sole source of phosphorus; however, the exact biochemistry of phosphonate degradation by this pathway is poorly understood. The protein was recombinantly expressed in Escherichia coli and purified to homogeneity. Sitting-drop vapour diffusion in combination with microseeding was used to obtain crystals that were suitable for X-ray diffraction. Data were collected to 1.3 A and the crystals belonged to space group C2, with unit-cell parameters a = 111.65, b = 75.41, c = 83.23 A, alpha = gamma = 90, beta = 126.3 degrees .