Friso van Assema

Contribution of the PD-1 ligands/PD-1 signaling pathway to dendritic cell-mediated CD4+ T cell activation.

Dendritic cells (DC) are extremely proficient inducers of naïve CD4+ T cell activation due to their high expression level of peptide‐MHC and an array of accessory molecules involved in cell migration, adhesion and co‐signaling, including PD‐1 ligand 1 (PD‐L1) and PD‐1 ligand 2 (PD‐L2). Whether PD‐L1 and PD‐L2 have a stimulatory or inhibitory function is a matter of debate, and could be partially dependent on the model system used. In this study we examined the role of PD‐L1 and PD‐L2 expressed by DC in naïve CD4+ T cell activation in a more physiologically relevant model system, using OVA‐specific T cells in combination with various levels of TCR stimulation. Overexpression of PD‐L1 or PD‐L2 by DC did not inhibit T cell proliferation, even when B7–1 and B7–2 mediated costimulation was absent, although IL‐2 production was consistently decreased. Surprisingly, blocking PD‐L1 and PD‐L2 with soluble programmed death‐1 (sPD‐1) also inhibited T cell activation, probably via reverse signaling via PD‐L1 and/or PD‐L2 into DC, leading to reduced DC maturation. This study suggests a relatively minor contribution of PD‐1 ligands in DC‐driven CD4+ T cell activation and provides evidence for reverse signaling by PD‐L1 and PD‐L2 into DC, resulting in a suppressive DC phenotype.

Asymmetric Synthesis of (S)-2-Indolinecarboxylic Acid by Combining Biocatalysis and Homogeneous Catalysis

(S)-2-Indolinecarboxylic acid, an intermediate for ACE inhibitors, was until recently produced by Fischer indole synthesis and classical resolution in seven steps. However, Perkin condensation to form ortho-chlorocinnamic acid, which is converted to (S)-ortho-chlorophenylalanine using the enzyme phenylalanine ammonia lyase prior to copper-catalyzed ring closure thereof delivers the enantiopure product in just three steps.

l-Selective Amidase with Extremely Broad Substrate Specificity from Ochrobactrum anthropi NCIMB 40321

ABSTRACT An industrially attractive l-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60°C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of α-hydrogen- and (bulky) α,α-disubstituted α-amino acid amides, α-hydroxy acid amides, and α-N-hydroxyamino acid amides. In all cases, only the l-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, β-amino and β-hydroxy acid amides, and dipeptides were not converted. The gene encoding this l-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.

The Crystal Structure of D-Threonine Aldolase from Alcaligenes xylosoxidans Provides Insight into a Metal Ion Assisted PLP-Dependent Mechanism

Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.