Identification of an acyl-enzyme intermediate in a meta-cleavage product hydrolase reveals the versatility of the catalytic triad

Meta-cleavage product (MCP) hydrolases are members of the α/β-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 ? resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of (18)O into the benzoate produced during hydrolysis in H(2)(18)O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES(red), previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.     

Mechanochemistry: opportunities for new and cleaner synthesis

The aim of this critical review is to provide a broad but digestible overview of mechanochemical synthesis, i.e. reactions conducted by grinding solid reactants together with no or minimal solvent. Although mechanochemistry has historically been a sideline approach to synthesis it may soon move into the mainstream because it is increasingly apparent that it can be practical, and even advantageous, and because of the opportunities it provides for developing more sustainable methods. Concentrating on recent advances, this article covers industrial aspects, inorganic materials, organic synthesis, cocrystallisation, pharmaceutical aspects, metal complexes (including metal-organic frameworks), supramolecular aspects and characterization methods. The historical development, mechanistic aspects, limitations and opportunities are also discussed (314 references).     

A restricted b‐spline basis for S‐shaped calibration curves

S‐shaped curves arise in many calibration assays. They are typically modeled using an empirically chosen nonlinear function. Nonparametric alternatives usually perform poorly because there are often insufficient data points and there are difficulties in enforcing the required asymptotic behavior. We propose an alternative approach using a b‐spline basis adapted to produce lower and upper asymptotes in the fitted curves. Its main advantage is that, with pre‐determined knot positions, the model is linear in the parameters and so is more numerically stable than a nonlinear model; this has advantages in, for example, fitting a mixed model to multiple curves. We explore the performance of this approach using data from a single radioimmunoassay for cortisol and from a set of 96 ELISA assays for the herbicide atrazine. Copyright © 2012 John Wiley & Sons, Ltd.     

Factoring multi-sublinear maps

Using Rademacher type, maximal estimates are established for k-sublinear operators with values in the space of measurable functions. Maurey–Nikishin factorization implies that such operators factor through a weak-type Lebesgue space. This extends known results for sublinear operators and improves some results for bilinear operators. For example, any continuous bilinear operator from a product of type 2 spaces into the space of measurable functions factors through a Banach space. Also included are applications for multilinear translation invariant operators.     

Specificity of the ester bond forming condensation enzyme SgcC5 in C-1027 biosynthesis

The SgcC5 condensation enzyme catalyzes the attachment of SgcC2-tethered (S)-3-chloro-5-hydroxy-β-tyrosine (2) to the enediyne core in C-1027 (1) biosynthesis. It is reported that SgcC5 (i) exhibits high stereospecificity toward the (S)-enantiomers of SgcC2-tethered β-tyrosine and analogues as donors, (ii) prefers the (R)-enantiomers of 1-phenyl-1,2-ethanediol (3) and analogues, mimicking the enediyne core, as acceptors, and (iii) can recognize a variety of donor and acceptor substrates to catalyze their regio- and stereospecific ester bond formations.     

The structure of the 3-separations of 3-connected matroids

Tutte defined a k-separation of a matroid M to be a partition (A,B) of the ground set of M such that |A|,|B|k and r(A)+r(B)−r(M)<k. If, for all m<n, the matroid M has no m-separations, then M is n-connected. Earlier, Whitney showed that (A,B) is a 1-separation of M if and only if A is a union of 2-connected components of M. When M is 2-connected, Cunningham and Edmonds gave a tree decomposition of M that displays all of its 2-separations. When M is 3-connected, this paper describes a tree decomposition of M that displays, up to a certain natural equivalence, all non-trivial 3-separations of M.     

First genetic analysis of Cryptosporidium from humans from Tasmania,and identification of a new genotype from a traveller to Bali

Little is known about the molecular composition of Cryptosporidium species from humans living in the insular state of Tasmania, Australia. In the present study, we genetically characterized 82 samples of Cryptosporidium from humans following conventional coproscopic testing in a routine, diagnostic laboratory. Using a PCR‐coupled single‐strand conformation polymorphism (SSCP) technique, targeting portions of the small subunit rRNA (SSU), and 60 kDa glycoprotein (gp60) loci, we identified two species of Cryptosporidium, including C. hominis (subgenotypes IbA10G2, IdA16, IeA12G3T3, and IfA19G1) and C. parvum (IIaA16G1R1 and IIaA18G3), and a new operational taxonomic unit (OTU) that genetically closely resembled C. wrairi. This OTU was further characterized using markers in the actin, Cryptosporidium oocyst wall protein (COWP), and 70 kDa heat shock protein (hsp70) genes. This study provides the first characterization of species and genotypes of Cryptosporidium from Tasmania, and presents clear genetic evidence, using five independent genetic loci, for a new genotype or species of Cryptosporidium in a Tasmanian person with a recent history of travelling to Bali, Indonesia. It would be interesting to undertake detailed molecular‐based studies of Cryptosporidium in Indonesia and neighbouring countries, in conjunction with morphological and experimental investigations of new genotypes.