The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

Recent studies have demonstrated that the O‐antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N‐formylated sugars (3‐formamido‐3,6‐dideoxy‐d ‐glucose or 4‐formamido‐4,6‐dideoxy‐d ‐glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6‐dehydratase, a pyridoxal 5'‐phosphate or PLP‐dependent aminotransferase, and an N‐formyltransferase. To date, there have been no published reports of N‐formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N‐formyltransferase. Given that M. tuberculosis produces l ‐rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6‐dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N‐formylated sugar in M. tuberculosis, namely a PLP‐dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.     

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

Campylobacter jejuni is a Gram‐negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain‐Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram‐negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O‐antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐glucose and dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5′‐phosphate dependent aminotransferase. Here, we describe the first three‐dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP‐3‐amino‐3,6‐dideoxy‐d ‐galactose or dTDP‐3‐amino‐3,6‐dideoxy‐d ‐glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C‐4′ carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution.     

Misannotations of the genes encoding sugar N‐formyltransferases

Tens of thousands of bacterial genome sequences are now known due to the development of rapid and inexpensive sequencing technologies. An important key in utilizing these vast amounts of data in a biologically meaningful way is to infer the function of the proteins encoded in the genomes via bioinformatics techniques. Whereas these approaches are absolutely critical to the annotation of gene function, there are still issues of misidentifications, which must be experimentally corrected. For example, many of the bacterial DNA sequences encoding sugar N‐formyltransferases have been annotated as l ‐methionyl‐tRNA transferases in the databases. These mistakes may be due in part to the fact that until recently the structures and functions of these enzymes were not well known. Herein we describe the misannotation of two genes, WP_088211966.1 and WP_096244125.1, from Shewanella spp. and Pseudomonas congelans, respectively. Although the proteins encoded by these genes were originally suggested to function as l ‐methionyl‐tRNA transferases, we demonstrate that they actually catalyze the conversion of dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose to dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose utilizing N¹⁰‐formyltetrahydrofolate as the carbon source. For this analysis, the genes encoding these enzymes were cloned and the corresponding proteins purified. X‐ray structures of the two proteins were determined to high resolution and kinetic analyses were conducted. Both enzymes display classical Michaelis–Menten kinetics and adopt the characteristic three‐dimensional structural fold previously observed for other sugar N‐formyltransferases. The results presented herein will aid in the future annotation of these fascinating enzymes.     

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC₅₀ of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.     

Three‐dimensional structure of a sugar N‐formyltransferase from Francisella tularensis

N‐formylated sugars have been observed on the O‐antigens of such pathogenic Gram‐negative bacteria as Campylobacter jejuni and Francisella tularensis. Until recently, however, little was known regarding the overall molecular architectures of the N‐formyltransferases that are required for the biosynthesis of these unusual sugars. Here we demonstrate that the protein encoded by the wbtj gene from F. tularensis is an N‐formyltransferase that functions on dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose as its substrate. The enzyme, hereafter referred to as WbtJ, demonstrates a strict requirement for N¹⁰‐formyltetrahydrofolate as its carbon source. In addition to the kinetic analysis, the three‐dimensional structure of the enzyme was solved in the presence of dTDP‐sugar ligands to a nominal resolution of 2.1 Å. Each subunit of the dimeric enzyme is dominated by a “core” domain defined by Met 1 to Ser 185. This core motif harbors the active site residues. Following the core domain, the last 56 residues fold into two α‐helices and a β‐hairpin motif. The hairpin motif is responsible primarily for the subunit:subunit interface, which is characterized by a rather hydrophobic pocket. From the study presented here, it is now known that WbtJ functions on C‐4′ amino sugars. Another enzyme recently investigated in the laboratory, WlaRD, formylates only C‐3′ amino sugars. Strikingly, the quaternary structures of WbtJ and WlaRD are remarkably different. In addition, there are several significant variations in the side chains that line their active site pockets, which may be important for substrate specificity. Details concerning the kinetic and structural properties of WbtJ are presented.     

Crystal structure of ADP‐dependent glucokinase from Methanocaldococcus jannaschii in complex with 5‐iodotubercidin reveals phosphoryl transfer mechanism

ADP‐dependent glucokinase (ADPGK) is an alternative novel glucose phosphorylating enzyme in a modified glycolysis pathway of hyperthermophilic Archaea. In contrast to classical ATP‐dependent hexokinases, ADPGK utilizes ADP as a phosphoryl group donor. Here, we present a crystal structure of archaeal ADPGK from Methanocaldococcus jannaschii in complex with an inhibitor, 5‐iodotubercidin, d ‐glucose, inorganic phosphate, and a magnesium ion. Detailed analysis of the architecture of the active site allowed for confirmation of the previously proposed phosphorylation mechanism and the crucial role of the invariant arginine residue (Arg197). The crystal structure shows how the phosphate ion, while mimicking a β‐phosphate group, is positioned in the proximity of the glucose moiety by arginine and the magnesium ion, thus providing novel insights into the mechanism of catalysis. In addition, we demonstrate that 5‐iodotubercidin inhibits human ADPGK‐dependent T cell activation‐induced reactive oxygen species (ROS) release and downstream gene expression, and as such it may serve as a model compound for further screening for hADPGK‐specific inhibitors.     

Structural studies on KijD1, a sugar C‐3′‐methyltransferase

Kijanimicin is an antitumor antibiotic isolated from Actinomadura kijaniata. It is composed of three distinct moieties: a pentacyclic core, a monosaccharide referred to as d ‐kijanose, and a tetrasaccharide chain composed of l ‐digitoxose units. d ‐Kijanose is a highly unusual nitro‐containing tetradeoxysugar, which requires at least ten enzymes for its production. Here we describe a structural analysis of one of these enzymes, namely KijD1, which functions as a C‐3′‐methyltransferase using S‐adenosylmethionine as its cofactor. For this investigation, two ternary complexes of KijD1, determined in the presence of S‐adenosylhomocysteine (SAH) and dTDP or SAH and dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐3‐methyl‐d ‐glucose, were solved to 1.7 or 1.6 Å resolution, respectively. Unexpectedly, these structures, as well as additional biochemical analyses, demonstrated that the quaternary structure of KijD1 is a dimer. Indeed, this is in sharp contrast to that previously observed for the sugar C‐3′‐methyltransferase isolated from Micromonospora chalcea. By the judicious use of site‐directed mutagenesis, it was possible to convert the dimeric form of KijD1 into a monomeric version. The quaternary structure of KijD1 could not have been deduced based solely on bioinformatics approaches, and thus this investigation highlights the continuing need for experimental validation.     

Probing the catalytic mechanism of a C-3'-methyltransferase involved in the biosynthesis of D-tetronitrose

D ‐Tetronitrose is a nitro‐containing tetradeoxysugar found attached to the antitumor and antibacterial agent tetrocarcin A. The biosynthesis of this highly unusual sugar in Micromonospora chalcea requires 10 enzymes. The fifth step in the pathway involves the transfer of a methyl group from S‐adenosyl‐L ‐methionine (SAM) to the C‐3′ carbon of dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐D ‐glucose. The enzyme responsible for this transformation is referred to as TcaB9. It is a monomeric enzyme with a molecular architecture based around three domains. The N‐terminal motif contains a binding site for a structural zinc ion. The middle‐ and C‐terminal domains serve to anchor the SAM and dTDP–sugar ligands, respectively, to the protein, and the active site of TcaB9 is wedged between these two regions. For this investigation, the roles of Tyr 76, His 181, Tyr 222, Glu 224, and His 225, which form the active site of TcaB9, were probed by site‐directed mutagenesis, kinetic analyses, and X‐ray structural studies. In addition, two ternary complexes of the enzyme with bound S‐adenosyl‐L ‐homocysteine and either dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐D ‐glucose or dTDP‐3‐amino‐2,3,6‐trideoxy‐D ‐galactose were determined to 1.5 or 1.6 Å resolution, respectively. Taken together, these investigations highlight the important role of His 225 in methyl transfer. In addition, the structural data suggest that the methylation reaction occurs via retention of configuration about the C‐3′ carbon of the sugar.     

Structural studies on the enzyme complex isopropylmalate isomerase (LeuCD) from Mycobacterium tuberculosis

The absence of the leucine biosynthesis pathway in humans makes the enzymes of this pathway in pathogenic bacteria such as Mycobacterium tuberculosis potential candidates for developing novel antibacterial drugs. One of these enzymes is isopropylmalate isomerase (IPMI). IPMI exists as a complex of two subunits: the large (LeuC) and the small (LeuD) subunit. The functional LeuCD complex catalyzes the stereospecific conversion reaction of α‐isopropylmalate to β‐isopropylmalate. Three C‐terminally truncated variants of LeuD have been analyzed by X‐ray crystallography to resolutions of 2.0 Å (LeuD_1–156), 1.2 Å (LeuD_1–168), and 2.5 Å (LeuD_1–186), respectively. The two most flexible parts of the structure are the regions of residues 30–37, the substrate discriminating loop, and of residues 70–74, the substrate binding loop. The three determined structures were also compared with the structures of other bacterial LeuDs. This comparison suggests the presence of two LeuD subfamilies. A model for the structure of the inactive enzyme complex has been obtained from solution X‐ray scattering experiments. The crystal structure of LeuD was shown to be compatible with the solution X‐ray scattering data from the small subunit. In contrast, the solution scattering results suggest that the large subunit LeuC and the LeuCD complex have overall shapes, which are radically different from the ones observed in the crystals of the functional homolog mitochondrial aconitase. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structure of decaprenylphosphoryl‐β‐ D‐ribose 2'‐epimerase from Mycobacterium smegmatis

Decaprenylphosphoryl‐β‐D ‐ribose 2'‐epimerase (DprE1) is an essential enzyme in the biosynthesis of cell wall components and a target for development of anti‐tuberculosis drugs. We determined the crystal structure of a truncated form of DprE1 from Mycobacterium smegmatis in two crystal forms to up to 2.35 Å resolution. The structure extends from residue 75 to the C‐terminus and shares homology with FAD‐dependent oxidoreductases of the vanillyl‐alcohol oxidase family including the DprE1 homologue from M. tuberculosis. The M. smegmatis DprE1 structure reported here provides further insights into the active site geometry of this tuberculosis drug target. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

A review on anti‐tuberculosis peptides: Impact of peptide structure on anti‐tuberculosis activity

Antibiotic resistance is a major public health problem globally. Particularly concerning amongst drug‐resistant human pathogens is Mycobacterium tuberculosis that causes the deadly infectious tuberculosis (TB) disease. Significant issues associated with current treatment options for drug‐resistant TB and the high rate of mortality from the disease makes the development of novel treatment options against this pathogen an urgent need. Antimicrobial peptides are part of innate immunity in all forms of life and could provide a potential solution against drug‐resistant TB. This review is a critical analysis of antimicrobial peptides that are reported to be active against the M tuberculosis complex exclusively. However, activity on non‐TB strains such as Mycobacterium avium and Mycobacterium intracellulare, whenever available, have been included at appropriate sections for these anti‐TB peptides. Natural and synthetic antimicrobial peptides of diverse sequences, along with their chemical structures, are presented, discussed, and correlated to their observed antimycobacterial activities. Critical analyses of the structure allied to the anti‐mycobacterial activity have allowed us to draw important conclusions and ideas for research and development on these promising molecules to realise their full potential. Even though the review is focussed on peptides, we have briefly summarised the structures and potency of the various small molecule drugs that are available and under development, for TB treatment.     

Potential anti‐bacterial drug target: Structural characterization of 3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase from Salmonella typhimurium LT2

3,4‐Dihydroxy‐2‐butanone‐4‐phosphate synthase (DHBPS) encoded by ribB gene is one of the first enzymes in riboflavin biosynthesis pathway and catalyzes the conversion of ribulose‐5‐phosphate (Ru5P) to 3,4‐dihydroxy‐2‐butanone‐4‐phosphate and formate. DHBPS is an attractive target for developing anti‐bacterial drugs as this enzyme is essential for pathogens, but absent in humans. The recombinant DHBPS enzyme of Salmonella requires magnesium ion for its activity and catalyzes the formation of 3,4‐dihydroxy‐2‐butanone‐4‐phosphate from Ru5P at a rate of 199 nmol min^?1 mg^?1 with K_m value of 116 μM at 37°C. Further, we have determined the crystal structures of Salmonella DHBPS in complex with sulfate, Ru5P and sulfate‐zinc ion at a resolution of 2.80, 2.52, and 1.86 Å, respectively. Analysis of these crystal structures reveals that the acidic loop (residues 34–39) responsible for the acid‐base catalysis is disordered in the absence of substrate or metal ion at the active site. Upon binding either substrate or sulfate and metal ions, the acidic loop becomes stabilized, adopts a closed conformation and interacts with the substrate. Our structure for the first time reveals that binding of substrate Ru5P alone is sufficient for the stabilization of the acidic active site loop into a closed conformation. In addition, the Glu38 residue from the acidic active site loop undergoes a conformational change upon Ru5P binding, which helps in positioning the second metal ion that stabilizes the Ru5P and the reaction intermediates. This is the first structural report of DHBPS in complex with either substrate or metal ion from any eubacteria. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structure of a putative pyridoxine 5′‐phosphate oxidase (Rv2607) from Mycobacterium tuberculosis

The three‐dimensional structure of Rv2607, a putative pyridoxine 5′‐phosphate oxidase (PNPOx) from Mycobacterium tuberculosis, has been determined by X‐ray crystallography to 2.5 Å resolution. Rv2607 has a core domain similar to known PNPOx structures with a flavin mononucleotide (FMN) cofactor. Electron density for two FMN at the dimer interface is weak despite the bright yellow color of the protein solution and crystal. The shape and size of the putative binding pocket is markedly different from that of members of the PNPOx family, which may indicate some significant changes in the FMN binding mode of this protein relative to members of the family. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

Active site conformational changes upon reaction intermediate biotinyl‐5'‐AMP binding in biotin protein ligase from Mycobacterium tuberculosis

Protein biotinylation, a rare form of post‐translational modification, is found in enzymes required for lipid biosynthesis. In mycobacteria, this process is essential for the formation of their complex and distinct cell wall and has become a focal point of drug discovery approaches. The enzyme responsible for this process, biotin protein ligase, substantially varies in different species in terms of overall structural organization, regulation of function and substrate specificity. To advance the understanding of the molecular mechanism of biotinylation in Mycobacterium tuberculosis we have biochemically and structurally characterized the corresponding enzyme. We report the high‐resolution crystal structures of the apo‐form and reaction intermediate biotinyl‐5'‐AMP‐bound form of M. tuberculosis biotin protein ligase. Binding of the reaction intermediate leads to clear disorder‐to‐order transitions. We show that a conserved lysine, Lys138, in the active site is essential for biotinylation.     

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

Pantoea ananatis is a Gram‐negative bacterium first recognized in 1928 as the causative agent of pineapple rot in the Philippines. Since then various strains of the organism have been implicated in the devastation of agriculturally important crops. Some strains, however, have been shown to function as non‐pathogenic plant growth promoting organisms. To date, the factors that determine pathogenicity or lack thereof between the various strains are not well understood. All P. ananatis strains contain lipopolysaccharides, which differ with respect to the identities of their associated sugars. Given our research interest on the presence of the unusual sugar, 4‐formamido‐4,6‐dideoxy‐d ‐glucose, found on the lipopolysaccharides of Campylobacter jejuni and Francisella tularensis, we were curious as to whether other bacteria have the appropriate biosynthetic machinery to produce these unique carbohydrates. Four enzymes are typically required for their biosynthesis: a thymidylyltransferase, a 4,6‐dehydratase, an aminotransferase, and an N‐formyltransferase. Here, we report that the gene SAMN03097714_1080 from the P. ananatis strain NFR11 does, indeed, encode for an N‐formyltransferase, hereafter referred to as PA1080c. Our kinetic analysis demonstrates that PA1080c displays classical Michaelis–Menten kinetics with dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose as the substrate and N¹⁰‐formyltetrahydrofolate as the carbon source. In addition, the X‐ray structure of PA1080c, determined to 1.7 Å resolution, shows that the enzyme adopts the molecular architecture observed for other sugar N‐formyltransferases. Analysis of the P. ananatis NFR11 genome suggests that the three other enzymes necessary for N‐formylated sugar biosynthesis are also present. Intriguingly, those strains of P. ananatis that are non‐pathogenic apparently do not contain these genes.     

Molecular architecture of KedS8, a sugar N‐methyltransferase from Streptoalloteichus sp. ATCC 53650

Kedarcidin, produced by Streptoalloteichus sp. ATCC 53650, is a fascinating chromoprotein of 114 amino acid residues that displays both antibiotic and anticancer activity. The chromophore responsible for its chemotherapeutic activity is an ansa‐bridged enediyne with two attached sugars, l ‐mycarose, and l ‐kedarosamine. The biosynthesis of l ‐kedarosamine, a highly unusual trideoxysugar, is beginning to be revealed through bioinformatics approaches. One of the enzymes putatively involved in the production of this carbohydrate is referred to as KedS8. It has been proposed that KedS8 is an N‐methyltransferase that utilizes S‐adenosylmethionine as the methyl donor and a dTDP‐linked C‐4′ amino sugar as the substrate. Here we describe the three‐dimensional architecture of KedS8 in complex with S‐adenosylhomocysteine. The structure was solved to 2.0 Å resolution and refined to an overall R‐factor of 17.1%. Unlike that observed for other sugar N‐methyltransferases, KedS8 adopts a novel tetrameric quaternary structure due to the swapping of β‐strands at the N‐termini of its subunits. The structure presented here represents the first example of an N‐methyltransferase that functions on C‐4′ rather than C‐3′ amino sugars.     

The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA)

The synthesis of deoxy-thymidine di-phosphate (dTDP)-L-rhamnose, an important component of the cell wall of many microorganisms, is a target for therapeutic intervention. The first enzyme in the dTDP-L-rhamnose biosynthetic pathway is glucose-1-phosphate thymidylyltransferase (RmlA). RmlA is inhibited by dTDP-L-rhamnose thereby regulating L-rhamnose production in bacteria. The structure of Pseudomonas aeruginosa RmlA has been solved to 1.66 A resolution. RmlA is a homotetramer, with the monomer consisting of three functional subdomains. The sugar binding and dimerization subdomains are unique to RmlA-like enzymes. The sequence of the core subdomain is found not only in sugar nucleotidyltransferases but also in other nucleotidyltransferases. The structures of five distinct enzyme substrate- product complexes reveal the enzyme mechanism that involves precise positioning of the nucleophile and activation of the electrophile. All the key residues are within the core subdomain, suggesting that the basic mechanism is found in many nucleotidyltransferases. The dTDP-L-rhamnose complex identifies how the protein is controlled by its natural inhibitor. This work provides a platform for the design of novel drugs against pathogenic bacteria.     

Two‐Photon polymerization for microfabrication of three‐dimensional scaffolds for tissue engineering application

In natural tissues cells are embedded in a three‐dimensional fibrous network of biopolymers like collagen, hyaluronic acid etc. This extracellular matrix (ECM) influences the cell fate, the differentiation status, metabolic processes and provides structural integrity. For a three‐dimensional or physiological cell cultivation that are required in biomedical applications (e.g. tissue engineering, BioMEMS) scaffolds are needed. These scaffolds mimic the ECM according to their biocompatibility which comprises aspects of surface compatibility and importantly for tissue engineering applications aspects of structural compatibility. We have evaluated scaffold design parameters for the three‐dimensional cultivation of chondrocytes for the tissue engineering of artificial cartilage. Two‐photon polymerization is a powerful technique for fabrication of polymeric three‐dimensional micro‐ and submicro‐structures. The photoinitiation system for two‐photon polymerization is excited by simultaneous absorption of two photons leading to chemical polymerization reactions. Due to a tight confinement of the excitation volume around the focal point, this method can produce micrometer sized objects maintaining a high spatial resolution down to 100 nm. Two‐photon processes require very high photon densities which are provided by pulsed femtosecond lasers. The potential of this approach for microfabrication of scaffolds for tissue engineering is demonstrated by investigation of the cell response to microstructures with complex three‐dimensional geometry and feature sizes in the range of few micrometers.