25‐Hydroxyvitamin D3 Synthesis by Enzymatic Steroid Side‐Chain Hydroxylation with Water

The hydroxylation of vitamin D₃ (VD₃, cholecalciferol) side chains to give 25‐hydroxyvitamin D₃ (25OHVD₃) is a crucial reaction in the formation of the circulating and biologically active forms of VD₃. It is usually catalyzed by cytochrome P450 monooxygenases that depend on complex electron donor systems. Cell‐free extracts and a purified Mo enzyme from a bacterium anaerobically grown with cholesterol were employed for the regioselective, ferricyanide‐dependent hydroxylation of VD₃ and proVD₃ (7‐dehydrocholesterol) into the corresponding tertiary alcohols with greater than 99 % yield. Hydroxylation of VD₃ strictly depends on a cyclodextrin‐assisted isomerization of VD₃ into preVD₃, the actual enzymatic substrate. This facile and robust method developed for 25OHVD₃ synthesis is a novel example for the concept of substrate‐engineered catalysis and offers an attractive alternative to chemical or O₂ /electron‐donor‐dependent enzymatic procedures.     

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

We analyzed the metal-binding properties of human centrin-2 (HsCen-2) and followed the changes in HsCen-2 structure upon metal-binding using micro-electrospray ionization mass spectrometry (muESI-MS). Apo-HsCen-2 is mostly monomeric. The ESI spectra of HsCen-2 show two charge-state distributions, representing two conformations of the protein. HsCen-2 binds four moles calcium/mol protein: one mol of calcium with high affinity, one additional mol of calcium with lower affinity, and two moles of calcium at low affinity sites. HsCen-2 binds four moles of magnesium/mol protein. The conformation giving the lower charge-state HsCen-2 by ESI, binds calcium and magnesium more readily than does the higher charge-state HsCen-2. Both conformations of HsCen-2 bind calcium more readily than magnesium. Calcium was more effective in displacing magnesium bound to HsCen-2 than vice versa. Binding of a peptide from a known binding partner, the xeroderma pigmentosum complementation group protein C (XPC), to apo-HsCen-2, occurs in the presence or the absence of calcium. Near and far-UV CD spectra of HsCen-2 show little difference with addition of calcium or magnesium. Minor changes in secondary structure are noted. Melting curves derived from temperature dependence of molar ellipticity at 222 nm for HsCen-2 show that calcium increases protein stability whereas magnesium does not. Delta 25 HsCen-2 behaves similarly to HsCen-2. We conclude that HsCen-2 binds calcium and magnesium and that calcium modulates HsCen-2 structure and function by increasing its stability without undergoing significant changes in secondary or tertiary structure.     

Actions of Commutative Hopf Algebras

We show that actions of finite-dimensional semisimple commutativeHopf algebras H on H-module algebras A are essentially group-gradings.Moreover we show that the centralizer of H in the smash productA # H equals A^H H. Using these we invoke results about groupgraded algebras and results about centralizers of separablesubalgebras to give connections between the ideal structureof A, A^H and A # H. Examples of the above occur naturally when one considers: (1) finite abelian groups G of automorphisms of an algebra Awith | G |^–1 A; (2) G-graded algebras, for finite groups G; (3) finite-dimensional restricted Lie algebras L, with semisimplerestricted enveloping algebra u(L), acting as derivations onan algebra A.     

Reactions of M(CO)5X (M = Mn, Re; X = Cl, Br) with (Ph2PCH2)3CCH3 (P3) and (Ph2P(CH2)2)3P (P3P'): synthetic, spectroscopic, electrochemical, and electrospray mass spectrometric studies

The reactions of M(CO)5X (M = Mn, Re; X = Cl, Br) with (Ph2PCH2)3CCH3 (P3) and (Ph2P(CH2)2)3P (P3P') are investigated, and the products are characterized by IR, NMR (31P and 13C), and electrospray mass spectrometric (ESMS) techniques. With P3, the major products are fac-M(CO)3(eta 2-P3)X (syn and anti isomers) and cis,fac-M(CO)2(eta 3-P3)X, and with P3P', the major product for each metal is cis,mer-M(CO)2(eta 3-P3P')X, but cis-[M(CO)2(eta 4-P3P')]X and fac-[Re(CO)3(eta 3-P3P')]X are also characterized. Addition of MeI to those complexes containing pendant phosphine groups produces the corresponding phosphonium cations without affecting the remainder of the molecule. On the voltammetric time scale, electrochemical oxidation of cis,fac-Mn(CO)2(eta 3-P3)X yields the corresponding 17e cation cis,fac-[Mn(CO)2(eta 3-P3)X]+, but on the longer time scale of exhaustive electrolysis or chemical oxidation, the product is fac-[Mn(CO)3(eta 3-P3)]+. In contrast, the rhenium cation cis,fac-[Re(CO)2(eta 3-P3)X]+ is stable on the synthetic time scale, but upon oxidation of cis,fac-Re(CO)2(eta 3-P3)X with NOBF4, the final product is the 18e [Re(CO)(NO)(eta 3-P3)X]+. cis,mer-Mn(CO)2(eta 3-P3P')X is reversibly oxidized to cis,mer-[Mn(CO)2(eta 3-P3P')X]+ on the voltammetric time scale, but on the longer synthetic time scale, the product isomerizes to trans-[Mn(CO)2(eta 3-P3P')X]+, which can be reduced to trans-Mn(CO)2(eta 3-P3P')X. Upon voltammetric oxidation, the corresponding rhenium complexes show an initial irreversible response associated with the pendant phosphine group prior to the reversible oxidation of the metal on the synthetic time scale; spectroscopic data indicate formation of cis,mer-Re(CO)2(eta 3-P3P'O)X. The complex cis,mer-[Re(CO)2(eta 3-P3P'Me)X]+ shows only the reversible metal oxidation response. ESMS data are obtained directly for the methylated cationic complexes, and neutral complexes are either oxidized or adducted with sodium ions to produce cationic species.     

Evidence for demethylation of syringyl moieties in archaeological wood using pyrolysis-gas chromatography/mass spectrometry

Archaeological oak (Quercus sp.) wood samples, ranging from 16(th) C. AD to 6000 BP, were studied using flash pyrolysis-gas chromatography/mass spectrometry to obtain insight into angiosperm lignin degradation. The pyrolysates revealed evidence of a number of 3-methoxy-1,2-benzenediol derivatives, methoxycatechols, directly related to 2,6-dimethoxyphenol, syringyl, moieties which are characteristic building blocks of angiosperm lignin. Mass spectra and mass chromatograms of these compounds are reported. The finding of these characteristic pyrolysis products in well-preserved archaeological wood provides unequivocal evidence that demethylation of syringyl units occurs very early in wood degradation. It is highly likely that the absence of abundant 3-methoxy-1, 2-benzenediols in degrading plant materials containing angiosperm lignin relates to the lability of these newly formed moieties.     

Comparison of methods for simultaneous identification of bacterial species and determination of metabolic activity by protein‐based stable isotope probing (Protein‐SIP) experiments

We developed a concept for analysing carbon and nitrogen fluxes in microbial communities by employing protein‐based stable isotope probing (Protein‐SIP) in metabolic labelling experiments with stable isotope labelled substrates. For identification of microbial species intact protein profiling (IPP) can be used, whereas the assessment of their metabolic activity is achieved by shotgun mass mapping (SMM). Microbial cultures were grown on substrates containing ¹³C or ¹⁵N. For identification of species we tested both the IPP and the SMM approaches. Mass spectra (MALDI‐MS) were taken from mixtures of either intact proteins or peptides from tryptic digestion for generating species‐specific peak patterns. In the case of SMM, the fragmentation of peptides was additionally used to obtain sequence information for species identification. Mass spectra of peptide sequences allow calculation of the amount of ¹³C or ¹⁵N incorporation within peptides for determining metabolic activity of the specific species. The comparison of IPP and SMM revealed a higher robustness of species identification by SMM. In addition, the assessment of incorporation levels of ¹³C and ¹⁵N into peptides by SMM revealed a lower uncertainty (0.5–0.8 atom %) compared to IPP (6.4–8.9 atom %). The determination of metabolic activity and function of individual species by Protein‐SIP can help to analyse carbon and nitrogen fluxes within microbial communities. Copyright © 2009 John Wiley & Sons, Ltd.     

The conformation of 4-phenylcyclohexanone in solution by 1H- and 13C-Lanthanide induced shift (L.I.S.) analysis : Evidence in favour of phenyl over t-butyl as a conformational lock

The degree of pucker of the cyclohexanone ring in 4-phenylcyclohexanone has been refined by L.I.S. analysis. The results indicate puckering intermediate between that in cyclohexanone and 4-t-butylcyclohexanone, suggesting that phenyl may be preferable to t-butyl as a locking group in conformational analysis. The Yb (fod)3 shift reagent, although binding primarily at the carbonyl oxygen atom, also complexes weakly with the phenyl group.     

Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy

The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) represents one of the hallmarks of Alzheimer's disease. We employed solid-state nuclear magnetic resonance (NMR) to investigate the structure and dynamics of PHFs formed in vitro by the three-repeat-domain (K19) of protein tau, representing the core of Alzheimer PHFs. While N and C termini of tau monomers in PHFs are highly dynamic and solvent-exposed, the rigid segment consists of three major beta-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited ((15)N, (13)C) and ((13)C, (13)C) correlation experiments suggests the existence of a fibril core that is largely built by repeat unit R3, flanked by surface-exposed units R1 and R4. Solid-state NMR, circular dichroism, and the fibrillization behavior of a K19 mutant furthermore indicate that electrostatic interactions play a central role in stabilizing the K19 PHFs.     

Incorporation of carbon and nitrogen atoms into proteins measured by protein-based stable isotope probing (Protein-SIP)

The identification of metabolically active microbial key players is fundamental for understanding the structure and functions of contaminant-degrading communities. The metabolic activity can be analysed by feeding the microbial culture with stable-isotope-labelled substrates and subsequently tracing their incorporation into the biomass. In this paper we present a method which is able to detect the incorporation of stable isotopes from the substrate into the proteins of a benzene-metabolising microorganism. Pseudomonas putida strain ML2 was grown under aerobic conditions with the substrates (12)C-benzene, (13)C-benzene or (15)N-ammonium and (12)C-benzene. Proteins of these cultures were resolved by two-dimensional gel electrophoresis (2-DE) and corresponding protein spots were subjected to matrix-assisted laser ionization/desorption mass spectrometric (MALDI-MS) analysis. The proteins of the (12)C-sample were identified by peptide mass fingerprinting (PMF) as well as by tandem mass spectrometric (MS/MS) measurements. The (13)C- or (15)N-content of the peptides from the labelling experiments was determined by MALDI-MS/MS. The incorporation of heavy isotopes into the proteins from cultures grown on (13)C-benzene and (15)N-ammonium was determined based on the mass differences between labelled and non-labelled peptides as well as on the isotopic distribution of the y(1)-ion of arginine. The method we present here principally allows the unravelling of the carbon and nitrogen flow not only in pure cultures, but also in microbial communities consisting of many microbial species. Copyright (c) 2008 John Wiley & Sons, Ltd.