Iterative approach to computational enzyme design

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes

Asymmetric synthesis of α,β-diamino acid derivatives with an aziridine-, azetidine- and γ-lactone-skeleton via Mannich-type additions across α-chloro- N-sulfinylimines

The efficient asymmetric synthesis of new chiral γ-chloro-α,β-diamino acid derivatives via highly diastereoselective Mannich-type reactions of N-(diphenylmethylene) glycine esters across a chiral α-chloro-N-p-toluenesulfinylimine was developed. The influence of the base, LDA or LiHMDS, used for the formation of the glycine enolates, was of great importance for the anti-/syn-diastereoselectivity of the Mannich-type reaction. The γ-chloro-α,β-diamino acid derivatives proved to be excellent building blocks for ring closure towards optically pure anti- and syn-β,γ-aziridino-α-amino esters, and subsequent ring transformation into trans-3-aminoazetidine-2-carboxylic acid derivatives and α,β-diamino- γ-butyrolactones

Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models

The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate

Replacement of nitrite in meat products by natural bioactive compounds results in reduced exposure to N‐Nitroso compounds: the PHYTOME project

Scope: It has been proposed that endogenously form N‐nitroso compounds (NOCs) are partly responsible for the link between red meat consumption and colorectal cancer (CRC) risk. As nitrite has been indicated as critical factor in the formation of NOCs, the impact of replacing the additive sodium nitrite (E250) by botanical extracts in the PHYTOME project is evaluated. Method and Results: A human dietary intervention study is conducted in which healthy subjects consume 300 g of meat for 2 weeks, in subsequent order: conventional processed red meat, white meat, and processed red meat with standard or reduced levels of nitrite and added phytochemicals. Consumption of red meat products enriched with phytochemicals leads to a significant reduction in the faecal excretion of NOCs, as compared to traditionally processed red meat products. Gene expression changes identify cell proliferation as main affects molecular mechanism. High nitrate levels in drinking water in combination with processed red meat intake further stimulates NOC formation, an effect that could be mitigated by replacement of E250 by natural plant extracts. Conclusion: These findings suggest that addition of natural extracts to conventionally processed red meat products may help to reduce CRC risk, which is mechanistically support by gene expression analyses

Determination of the number of light neutrino species from single photon production at LEP

A determination of the number of light neutrino families performed by measuring the cross section of single photon production in \ee\ collision near the \Zo\ resonance is reported. From an integrated luminosity of $100~\mathrm{pb^{-1}}$, collected during the years 1991--94, we have observed 2091 single photon candidates with an energy above 1~\GeV\ in the polar angular region $45^\circ < \theta_\gamma < 135^\circ$. From a maximum likelihood fit to the single photon cross section, the \Zo\ decay width into invisible particles is measured to be \Ginv = 498 \pm 12 \mathrm{(stat)} \pm 12 \mathrm{(sys)~MeV}. Using the Standard Model couplings of neutrinos to the \Zo, the number of light neutrino species is determined to be $N_\nu = 2.98 \pm 0.07 (\mathrm{stat}) \pm 0.07 (\mathrm{sys}).

Search for R-Parity Breaking Sneutrino Exchange at LEP

We report on a search for R--parity breaking effects due to supersymmetric tau--sneutrino exchange in the reactions e+e- to e+e- and e+e- to mu+mu- at centre--of--mass energies from 91~{\GeV} to 172~{\GeV}, using the L3 detector at LEP. No evidence for deviations from the Standard Model expectations of the measured cross sections and forward--backward asymmetries for these reactions is found. Upper limits for the couplings $\lambda_{131}$ and $\lambda_{232}$ for sneutrino masses up to m_{\SNT} \leq 190~\GeV are determined from an analysis of the expected effects due to tau sneutrino exchange

Search for charged Higgs bosons in e+e−e^+ e^- collisions at centre-of-mass energies between 130 and 183 GeV

A search for pair-produced charged Higgs bosons is performed with the L3 detector at LEP using data collected at centre-of-mass energies from \mbox{130 to 183 \GeV{}}, corresponding to an integrated luminosity of 88.3 \pb. The Higgs decays into a charm and a strange quark or into a tau lepton and its associated neutrino are considered. The observed candidates are consistent with the expectations from Standard Model background processes. A lower limit of 57.5 \GeV{} on the charged Higgs mass is derived at 95\% CL, independent of the decay branching ratio \mathrm{Br(H^\pm\ra \tau\nu)}

Heavy Quarkonium Production in Z Decays

We report measurements of the inclusive production of heavy quarkonium states in $\mathrm {Z}$ decays based on the analysis of 3.6 million hadronic events collected by the L3 detector at LEP. The measurement of inclusive J production and an improved $95\%$ confidence level upper limit on $\Upsilon$ production are presented. In addition, two independent measurements of the ratio, $f_{\mathrm{p}}$, of prompt J mesons to those from B decay are made using two different isolation cuts to separate prompt J mesons from J mesons produced in the decays of b hadrons. The results are: % \begin{eqnarray} \mathrm{Br}(\mathrm{Z} \rightarrow \mathrm{J} + \mathrm{X}) & = & (3.21 \pm 0.21 \; \mathrm{(stat.)} \; ^{+ 0.19}_{- 0.28} \; \mathrm{(sys.)} ) \times 10^{-3} \; , \nonumber \\ \mathrm{Br}(\mathrm {Z} \rightarrow \Upsilon(\mathrm{1S} + X) & < & 4.4 \times 10^{-5} \; , \nonumber \\ %% f_{\mathrm{p}} & = & (7.1 \pm 2.1 \; \mathrm{(stat.)} \; \pm 1.2 \; \mathrm{(sys.)} \; ^{+1.5}_{-0.8} \;\mathrm{(theo.)} ) \times 10^{-2} \; . \nonumbe