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K Pfizenmaier

Publications and source records attributed to K Pfizenmaier.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cross-talk mechanisms in the development of insulin resistance of skeletal muscle cells palmitate rather than tumour necrosis factor inhibits insulin-dependent protein kinase B (PKB)/Akt stimulation and glucose uptake.

Insulin resistance in skeletal muscle is one of the earliest symptoms associated with non-insulin-dependent diabetes mellitus (NIDDM). Tumour necrosis factor (TNF) and nonesterified fatty acids have been proposed to be crucial factors in the development of the insulin-resistant state. We here show that, although TNF downregulated insulin-induced insulin receptor (IR) and IR substrate (IRS)-1 phosphorylation as well as phosphoinositide 3-kinase (PI3-kinase) activity in pmi28 myotubes, this was, unlike in adipocytes, not sufficient to affect insulin-induced glucose transport. Rather, TNF increased membrane expression of GLUT1 and glucose transport in these muscle cells. In contrast, the nonesterified fatty acid palmitate inhibited insulin-induced signalling cascades not only at the level of IR and IRS-1 phosphorylation, but also at the level protein kinase B (PKB/Akt), which is thought to be directly involved in the insulin-induced translocation of GLUT4, and inhibited insulin-induced glucose uptake. Palmitate also abrogated TNF-dependent enhancement of basal glucose uptake, suggesting that palmitate has the capacity to render muscle cells resistant not only to insulin but also to TNF with respect to glucose transport by GLUT4 and GLUT1, respectively. Our data illustrate the complexity of the mechanisms governing insulin resistance of skeletal muscle, questioning the role of TNF as a direct inhibitor of glucose homoeostasis in this tissue and shedding new light on an as yet unrecognized multifunctional role for the predominant nonesterified fatty acid palmitate in this process.

Animals↗
PubMed1999

Regulation of the murine NMDA-receptor-subunit NR2C promoter by Sp1 and fushi tarazu factor1 (FTZ-F1) homologues.

We have cloned the 5'-region of the murine N-methyl-d-aspartate (NMDA) receptor channel subunit NR2C (GluRepsilon3) gene and characterized the cis- and trans-activating regulatory elements responsible for its tissue specific activity. By using a native epsilon3-promoter/lacZ-construct & various 5'-deletion constructs, we compared beta-galactosidase expression in non-neuronal NIH3T3 cells and in neuronal epsilon3-gene-expressing HT-4 cells and show that large parts of the epsilon3 promoter are responsible for the repression of the epsilon3 gene in non-neuronal cells. Deletion of exon 1 sequences led to an enhancement of epsilon3 transcription, suggesting a role of the 5'-untranslated region in epsilon3 gene regulation. Sequence analysis of the promoter region revealed potential binding sites for the transcription factor Sp1, the murine fushi tarazu factor1 (FTZ-F1) homologues, embryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF-1), as well as for the chicken ovalbumin upstream promoter transcription-factor (COUP-TF). Electrophoretic mobility shift assays confirmed specific binding of Sp1, SF-1 and COUP-TFI. Whereas point mutation studies indicate that, in neuronal HT-4 cells, Sp1 is apparently not critically involved in basal epsilon3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of epsilon3-promoter-driven reporter gene expression upon cotransfection of an SF1-expression vector, which was reverted by deletion and point mutation of the SF1 binding site.

3T3 Cells↗
PubMed1999

Preferential expression of tumor necrosis factor receptor 55 (TNF-R55) on human articular chondrocytes: selective transcriptional upregulation of TNF-R75 by proinflammatory cytokines interleukin 1beta, tumor necrosis factor-alpha, and basis fibroblast growth factor.

OBJECTIVE: Articular cartilage is the main target for tumor necrosis factor-alpha (TNF-alpha) and interleukin 1(IL-1) actions. These cytokines are believed to mediate cartilage degradation in arthritis. We studied the expression of TNF receptors (TNF-R) on human articular chondrocytes and their regulation by IL-1beta, TNF-alpha, and basic fibroblast growth factor (bFGF). METHODS: The expression of TNF-R55 and TNF-R75 on human nonarthritic articular chondrocytes was analyzed on protein and mRNA levels by ligand binding studies and reverse transcription polymerase chain reaction (RT-PCR) technique. The regulation of these receptors induced by IL-1 TNF-alpha, and bFGF on mRNA level was studied using RT-PCR. RESULTS: Both TNF-R55 and TNF-R75 are expressed constitutively on human articular chondrocytes, and the number of both receptors varied between 822 and 3880 receptors per cell, depending on the donor cartilage used. Using TNF receptor-specific antibodies, we show that normal chondrocytes express mainly TNF-R55. These results are consistent with the mRNA data obtained by RT-PCR. mRNA expression of TNF receptors is regulated by IL-1beta, TNF-alpha, and bFGF. On human chondrocytes the expression of TNF-R75 mRNA was markedly upregulated by IL-ID, TNF-alpha, and bFGF, whereas the expression of TNF-R55 mRNA remained largely unchanged. A combination of IL-1beta and TNF-alpha, but not of IL-1beta and bFGF, showed an additive effect on TNF-R75 mRNA expression. CONCLUSION: The expression of TNF-R55 and TNF-R75 on human articular chondrocytes is modulated independently by IL-1beta, TNF-alpha, and bFGF, suggesting a role of these regulatory mechanisms in the degradation processes of human articular cartilage in inflammatory joint diseases.

Antigens, CD↗
PubMed1998

TNF inhibits insulin induced STAT5 activation in differentiated mouse muscle cells pmi28.

Tumor necrosis factor (TNF) plays a central role in the state of insulin resistance leading to type II diabetes. We here describe the crosstalk of TNF with insulin signaling cascades in the mouse muscle cell line pmi28. TNF downregulated insulin induced insulin receptor kinase activity and insulin induced activation of the transcription factor STAT5. Our results provide evidence that the inhibitory crosstalk between TNF and insulin in skeletal muscle cells comprises an interference with the expression of STAT5 regulated genes which may play an important role in the manifestation and/or progression of insulin resistance in muscle cells.

Animals↗
PubMed1998

Tumor necrosis factor induces ceramide oscillations and negatively controls sphingolipid synthases by caspases in apoptotic Kym-1 cells.

The role, origin, and mode of action of the lipid messenger ceramide in programmed cell death and its linkage to receptor-associated apoptotic signal proteins is still unresolved. We show here in Kym-1 rhabdomyosarcoma cells that tumor necrosis factor (TNF)-induced apoptosis is preceded by a multiphasic increase in intracellular ceramide levels. Distinct enzymes were found to contribute to three waves of ceramide, neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase, with peak activities at 1-2, 40, and around 200 min, respectively, the latter coinciding with progression to irreversible damage. In parallel with ceramide generation, TNF-mediated inhibition of glucosylceramide and sphingomyelin (SM) synthase prevents the immediate metabolization of this lipid mediator. In the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) or benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethyl ketone (Z-DEVD-cmk), a broad spectrum and a caspase 3-selective inhibitor, respectively, glucosylceramide and SM synthase activity remains unaffected by TNF, and intracellular ceramide accumulation is not observed. Our results show that several lipid enzymes contribute to generation of ceramide in response to TNF and identify glucosylceramide and SM synthase as important regulators of the kinetics and magnitude of intracellular ceramide accumulation. As glucosylceramide and SM synthase activity is caspase-sensitive, our data suggest a novel functional link between caspase(s) and ceramide during apoptotic processes.

4-Chloro-7-nitrobenzofurazan↗
PubMed1998

Monitoring of scFv selected by phage display using detection of scFv-pIII fusion proteins in a microtiter scale assay.

We describe here a method for the efficient and rapid analysis of antigen binding characteristics of recombinant antibodies (ab) selected by phage display. This novel approach combines the bacterial production of soluble single chain ab (scFv)-pIII fusion proteins on a microtiter scale with the detection of these fusion proteins via a pIII-specific ab. It facilitates the parallel analysis of large numbers of clones and is more efficient than current analysis protocols. Applying this technique, we analysed phage display selection of tetanus toxoid (TTX) specific scFv with respect to: (i) the productive expression of fusion proteins; (ii) the enrichment of specific scFv in subsequent rounds of phage display selection on a polyclonal level; (iii) the antigen specificity of individual scFv clones; (iv) the antigen binding affinity of a selected scFv. A TTX-specific scFv (clone 4.3) was further examined in a mono- and bivalent form by surface plasmon resonance analysis. ScFv 4.3 possesses a subnanomolar affinity and a low off rate constant.

Animals↗
PubMed1998

Protein kinase Cmu downregulation of tumor-necrosis-factor-induced apoptosis correlates with enhanced expression of nuclear-factor-kappaB-dependent protective genes.

Protein kinase Cmu (PKCmu) represents a new subtype of the PKC family characterized by the presence of a pleckstrin homology (PH) domain and an amino-terminal hydrophobic region. In order to analyse the potential role of PKCmu in signal-transduction pathways, stable PKCmu transfectants were established with human and murine cell lines. All transfectants showed a reduced sensitivity to tumor-necrosis-factor (TNF)-induced apoptosis, which correlated with the amount of transgene expressed and with an enhanced basal transcription rate of NF-kappaB-driven genes including the inhibitor of apoptosis protein 2 (cIAP2) and TNF-receptor-associated protein 1 (TRAF1). Sensitivity to apoptosis induced by the lipid mediator ceramide was unchanged in PKCmu transfectants. In support of a PKCmu action on NF-kappaB, we show enhancement and downregulation of TNF-induced expression of a NF-kappaB-dependent reporter gene by transient overexpression of wild-type and kinase-negative mutants of PKCmu, respectively. Interestingly, no significant changes were found in an electrophoretic mobility shift assay, indicative of PKCmu action downstream of IkappaB degradation, probably by modulation of the transactivation capacity of NF-kappaB. The dominant negative action of the kinase-negative mutant further suggest a regulatory role of PKCmu for NF-kappaB-dependent gene expression.

Apoptosis↗
PubMed1998

TNFR80-dependent enhancement of TNFR60-induced cell death is mediated by TNFR-associated factor 2 and is specific for TNFR60.

Costimulation of TNFR80 can strongly enhance TNFR60-induced cell death. In this study, we show that this enhancement is TNFR60 selective, as neither TNF-related apoptosis-inducing ligand/Apo2 ligand-, Apo1/Fas-, ceramide-, nor daunorubicin-mediated cell death was affected by costimulation of TNFR80. We further demonstrate that TNFR-associated factor 2 (TRAF2) is critically involved in both negative and positive regulation of TNF-induced cell death. Overexpression of TRAF2 and of a TRAF2 mutant, deficient in nuclear factor-kappaB activation, selectively desensitized and enhanced, respectively, TNFR60-induced cell death in HeLa cells. However, upon costimulation of TNFR80, which mediates activation of nuclear factor-kappaB and the c-Jun amino-terminal kinase via TRAF2, TNF-induced cell death is drastically enhanced in parental and TRAF2-transfected, but not in TRAF2 (87-501)-transfected cells. These data point to a critical role of TRAF2 in the apoptotic TNFR cross talk, whereby the TNFR80-dependent enhancement of TNFR60-induced cell death is due to TNFR80-mediated negative regulation of TRAF2 function(s). An interference with TRAF2 function was confirmed independently by analysis of c-Jun amino-terminal kinase activation via TNFR60 upon prestimulation of TNFR80. We propose that the apoptotic TNFR cross talk is based on TNFR80-mediated abrogation of antiapoptotic TRAF2-dependent signaling pathways initiated by TNFR60, but not Apo1/Fas or the apoptotic TNF-related apoptosis-inducing ligand receptors.

Adjuvants, Immunologic↗
PubMed1998

Cloning and characterization of promoter and 5'-UTR of the NMDA receptor subunit epsilon 2: evidence for alternative splicing of 5'-non-coding exon.

Using rapid amplification of cDNA ends (RACE), we have cloned the 5'-untranslated region (5'-UTR) of the N-methyl-D-aspartate receptor subunit epsilon 2 from murine forebrain-derived mRNA. We identified two distinct types of cDNA species differing in the presence or absence of one exon sequence. Sequencing of the 5'-non-coding region of the epsilon 2 gene revealed that the epsilon 2 5'-UTR consists of three untranslated exons located at least 20 kb upstream of exon 4 that contains the ATG codon for initiation of translation. This genomic organization shows a close similarity to the epsilon 3 gene. The transcriptional start site was determined by primer extension assays. Expression of the alternative exon sequence was shown by in situ hybridization in the murine brain. Basal transcriptional activity of the epsilon 2 promoter was detected in different neuronal and non-neuronal cell lines with transient reporter gene expression assays. Potential SP1 and CREB binding sites were found in the promoter region. Specific binding of these transcription factors was demonstrated in electrophoretic mobility shift assays.

3T3 Cells↗
PubMed1998

Regulation of Raf-1 kinase by TNF via its second messenger ceramide and cross-talk with mitogenic signalling.

Raf-1 kinase is a central regulator of mitogenic signal pathways, whereas its general role in signal transduction of tumour necrosis factor (TNF) is less well defined. We have investigated mechanisms of Raf-1 regulation by TNF and its messenger ceramide in cell-free assays, insect and mammalian cell lines. In vitro, ceramide specifically bound to the purified catalytic domain and enhanced association with activated Ras proteins, but did not affect the kinase activity of Raf-1. Cell-permeable ceramides induced a marked increase of Ras-Raf-1 complexes in cells co-expressing Raf-1 and activated Ras. Likewise, a fast elevation of the endogeneous ceramide level, induced by TNF treatment of human Kym-1 rhabdomyosarcoma cells, was followed by stimulation of Ras-Raf-1 association without significant Raf-1 kinase activation. Failure of TNF or ceramide to induce Raf-1 kinase was observed in several TNF-responsive cell lines. Both TNF and exogeneous C6-ceramide interfered with the mitogenic activation of Raf-1 and ERK by epidermal growth factor and down-regulated v-Src-induced Raf-1 kinase activity. TNF also induced the translocation of Raf-1 from the cytosolic to the particulate fraction, indicating that this negative regulatory cross-talk occurs at the cell membrane. Interference with mitogenic signals at the level of Raf-1 could be an important initial step in TNF's cytostatic action.

3T3 Cells↗
PubMed1998

Guided selection of antibody fragments specific for human interferon gamma receptor 1 from a human VH- and VL-gene repertoire.

OBJECTIVE: The guided selection strategy for isolation of human antibody (Ab) fragments specific for human interferon gamma receptor 1 (IFNGR-1) from a cloned Ab VH and VL repertoire has been investigated. In order to identify recombinant Abs binding to soluble antigen, a novel method termed affinity sedimentation was introduced here. RESULTS AND CONCLUSIONS: The VH region of murine monoclonal Ab (IR gamma-1) against human IFNGR-1 was combined with human VL repertoire and used for selection of human VL regions. One of these human VL regions (kappa 2) possesses high homology to the murine template VL region, also in CDR3 (77%). A chimeric Fab consisting of kappa 2 and the murine IR gamma-1 VH region was highly IFNGR-1 specific and exerted the same epitope specificity and a comparable binding affinity as the parental murine Fab. In a further step, the selected human VL region kappa 2 was combined with a human VH repertoire and led by guided selection to the generation of a completely human Fab (1b5) specific for human IFNGR-1. The overall VH region homology of 1b5 compared to the parental antibody IR gamma-1 was 81%, with a rather low homology in CDR3. Binding competition studies revealed that the epitope recognized by 1b5 differs from the parental Ab IR gamma-1.

Amino Acid Sequence↗
PubMed1998

Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli.

Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

Animals↗
PubMed1997

Peyer's patch organogenesis is intact yet formation of B lymphocyte follicles is defective in peripheral lymphoid organs of mice deficient for tumor necrosis factor and its 55-kDa receptor.

Targeted inactivation of genes in the tumor necrosis factor (TNF)/lymphotoxin (LT) ligand and receptor system has recently revealed essential roles for these molecules in lymphoid tissue development and organization. Lymphotoxin-alphabeta (LTalphabeta)/lymphotoxin-beta receptor (LTbeta-R) signaling is critical for the organogenesis of lymph nodes and Peyer's patches and for the structural compartmentalization of the splenic white pulp into distinct B and T cell areas and marginal zones. Moreover, an essential role has been demonstrated for TNF/p55 tumor necrosis factor receptor (p55TNF-R) signaling in the formation of splenic B lymphocyte follicles, follicular dendritic cell networks, and germinal centers. In contrast to a previously described essential role for the p55TNF-R in Peyer's patch organogenesis, we show in this report that Peyer's patches are present in both TNF and p55TNF-R knockout mice, demonstrating that these molecules are not essential for the organogenesis of this lymphoid organ. Furthermore, we show that in the absence of TNF/p55TNF-R signaling, lymphocytes segregate normally into T and B cell areas and a normal content and localization of dendritic cells is observed in both lymph nodes and Peyer's patches. However, although B cells are found to home normally within Peyer's patches and in the outer cortex area of lymph nodes, organized follicular structures and follicular dendritic cell networks fail to form. These results show that in contrast to LTalphabeta signaling, TNF signaling through the p55TNF-R is not essential for lymphoid organogenesis but rather for interactions that determine the cellular and structural organization of B cell follicles in all secondary lymphoid tissues.

Animals↗
PubMed1997

Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells.

Tumor necrosis factor (TNF) and lymphotoxin (LT) are highly pleiotropic cytokines that play a central role in regulating HIV-1 replication. These cytokines express their activities through two membrane receptors, TNFR60 (p55-60) and TNFR80 (p75-80). In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production. Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells. More relevant, in vitro HIV-infected peripheral blood lymphocytes cocultured with cells expressing membrane TNF underwent rapid induction of apoptosis with a subsequent reduced HIV production of these lymphocytes cultures. This was not observed with HIV-infected lymphocytes treated with soluble TNF. These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

Acquired Immunodeficiency Syndrome↗
PubMed1997

In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis.

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)-induced, CD4+ T cell-dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A-induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild-type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.

Animals↗
PubMed1997

A TNF receptor antagonistic scFv, which is not secreted in mammalian cells, is expressed as a soluble mono- and bivalent scFv derivative in insect cells.

Single chain antibodies (scFv) are usually produced in E. coli, but generation of certain scFv derivatives, such as complex fusion proteins or glycosylated forms of scFv is restricted to eukaryotic expression systems. We investigated the production of soluble mono- and bivalent single chain antibodies (scFv) in eukaryotic cells and describe a cassette vector system for mammalian and baculovirus expression which is compatible with an established vector system for bacterial expression and phage display selection of scFvs. The applied model scFv was derived from a murine antibody (H398) against human tumor necrosis factor receptor 1 (TNFR60), known to be a potent antagonist of TNF action in its monomeric form and a potential therapeutic agent for treatment of TNF-mediated diseases. Surprisingly, the monomeric scFv form of H398 (scFv H398) is expressed but not secreted in different mammalian cells. In contrast, in insect cells using recombinant baculovirus, a monovalent scFv H398 and a bivalent scFv fusion protein with an human IgG1 Fc region were expressed and secreted with correctly processed signal sequence. Concerning the influence of valency of the model Ab and its derivatives on antigen binding affinity and neutralisation of TNF activity, we found that the mono- and bivalent form of scFv H398 possesses the same characteristics as proteolytically produced Fab H398 and original mAb H398, respectively. Furthermore, fusion of the Ig Fc protein to scFv H398 increase the in vitro half-life at 37 degrees C. We conclude that the described cassette vectors readily allow the eukaryotic expression of mono- and bivalent scFv derivatives to analyse the influence of valency of scFv molecules on antigen binding and biological activity.

Animals↗
PubMed1996

Identification of potential active-site residues in the hydroxynitrile lyase from Manihot esculenta by site-directed mutagenesis.

The hydroxynitrile lyase from cassava (Manihot esculenta Crantz) (EC 4.1.2.37) catalyzes the decomposition of the achiral alpha-hydroxynitrile acetone cyanohydrin into HCN and acetone during cyanogenesis of damaged plants. This enzyme can also be used for stereoselective synthesis of a wide array of (S)-cyanohydrins by addition of HCN to aldehydes or ketones. Optically active cyanohydrins are interesting intermediates for the synthesis of alpha-hydroxy acids, alpha-hydroxy ketones, or beta-ethanolamines, all of which are important building blocks in organic synthesis. Inhibition of hydroxynitrile lyase from M. esculenta (MeHNL) by serine- and histidine-modifying reagents suggests involvement of active site seryl and histidyl residues. Furthermore, serine 80 of MeHNL is part of the active site motif Gly-X-Ser-X-Gly/Ala, often considered as the hallmark of catalytic triads having independently evolved in four groups of enzymes: the alpha/beta hydrolase fold enzymes, subtilisins, the cysteine proteases, and the eukaryotic serine proteases. By site-directed mutagenesis, three residues critical for enzyme activity have been identified: serine 80, aspartic acid 208, and histidine 236. These residues may be directly involved in MeHNL-catalyzed decomposition of cyanohydrins, providing evidence for a catalytical triad in HNLs, too. The order of the catalytic triad residues in the primary sequence of MeHNL is nucleophile-histidine-acid, suggesting that MeHNL belongs to the alpha/beta hydrolase fold group of enzymes. In contrast to all other enzymes having a catalytical triad, HNLs catalyze no net hydrolytic reactions.

Aldehyde-Lyases↗