Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E.

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min-1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

Immobilized Dipeptidase in Manganese Ion-Loaded Polyethylenimine-Induced Calcium Phosphate Nanocrystals for Carnosine Synthesis.

Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.

Prolidase activity assays. A survey of the reported literature methodologies.

Prolidase (EC.3.4.13.9) is a dipeptidase known nowadays to play a pivotal role in several physiological and pathological processes. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), thus finely regulating the homeostasis of free proline and hydroxyproline. Abnormally high or low levels of prolidase have been found in numerous acute and chronic syndromes affecting humans (chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection, and others) for which the content of proline is well recognized as a clinical marker. As a consequence, the accurate analytical determination of prolidase activity is of greatly significant importance in clinical diagnosis and therapy. Apart from the Chinard's assay, some other more sensitive and well validated methodologies have been published. These include colorimetric and spectrophotometric determinations of free proline produced by enzymatic reactions, capillary electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, electrochemoluminescence, thin layer chromatography, and HPLC. The aim of this comprehensive review is to make a detailed survey of the in so far reported analytical techniques, highlighting their general features, as well as their advantages and possible drawbacks, providing in the meantime suggestions to stimulate further research in this intriguing field.

Human carnosinases: A brief history, medicinal relevance, and in silico analyses.

Carnosine, an endogenous dipeptide, has been found to have a plethora of medicinal properties, such as antioxidant, antiageing, and chelating effects, but with one downside: a short half-life. Carnosinases and two hydrolytic enzymes, which remain enigmatic, are responsible for these features. Hence, here we emphasize why research is valuable for better understanding crucial concepts like ageing, neurodegradation, and cancerogenesis, given that inhibition of carnosinases might significantly prolong carnosine bioavailability and allow its further use in medicine. Herein, we explore the literature regarding carnosinases and present a short in silico analysis aimed at elucidating the possible recognition pattern between CN1 and its ligands.

The amino-dipeptidyl peptidases DPP8 and DPP9: Purification and enzymatic assays.

Proline residues highly impact protein stability when present either in the first or second N-terminal position. While the human genome encodes for more than 500 proteases, only few proteases are capable of hydrolyzing a proline-containing peptide bond. The two intra-cellular amino-dipeptidyl peptidases DPP8 and DPP9 are exceptional as they possess the rare ability to cleave post-proline. By removing N-terminal Xaa-Pro dipeptides, DPP8 and DPP9 expose a neo N-terminus of their substates, which can consequently alter inter- or intra-molecular interactions of the modified protein. Both DPP8 and DPP9 play key roles in the immune response and are linked to cancer progression, emerging as attractive drug targets. DPP9 is more abundant than DPP8 and is rate limiting for cleavage of cytosolic proline-containing peptides. Only few DPP9 substrates have been characterized; these include Syk, a central kinase for B-cell receptor mediated signaling; Adenylate Kinase 2 (AK2) which is important for cellular energy homeostasis; and the tumor suppressor Breast cancer type 2 susceptibility protein (BRCA2) that is critical for repair of DNA double strand breaks. N-terminal processing of these proteins by DPP9 triggers their rapid turn-over by the proteasome, highlighting a role for DPP9 as upstream components of the N-degron pathway. Whether N-terminal processing by DPP9 leads to substrate-degradation in all cases, or whether additional outcomes are possible, remains to be tested. In this chapter we will describe methods for purification of DPP8 and DPP9 as well as protocols for biochemical and enzymatic characterization of these proteases.

Mechanistic insights on anserine hydrolyzing activities of human carnosinases.

Anserine and carnosine represent histidine-containing dipeptides that exert a pluripotent protective effect on human physiology. Anserine is known to protect against oxidative stress in diabetes and cardiovascular diseases. Human carnosinases (CN1 and CN2) are dipeptidases involved in the homeostasis of carnosine. In poikilothermic vertebrates, the anserinase enzyme is responsible for hydrolyzing anserine. However, there is no specific anserine hydrolyzing enzyme present in humans. In this study, we have systematically investigated the anserine hydrolyzing activity of human CN1 and CN2. A targeted multiple reaction monitoring (MRM) based approach was employed for studying the enzyme kinetics of CN1 and CN2 using carnosine and anserine as substrates. Surprisingly, both CN1 and CN2 can hydrolyze anserine effectively. The observed catalytic turnover rate (Vmax/[E]t) was 21.6 s-1 and 2.8 s-1 for CN1 and CN2, respectively. CN1 is almost eight-fold more efficient in hydrolyzing anserine compared to CN2, which is comparable to the efficiency of the carnosine hydrolyzing activity of CN2. The Michaelis constant (Km) value for CN1 (1.96 mM) is almost three-fold lower compared to CN2 (6.33 mM), representing higher substrate affinity for anserine-CN1 interactions. Molecular docking studies showed that anserine binds at the catalytic site of the carnosinases with an affinity similar to carnosine. Overall, the present study elucidated the inherent promiscuity of human carnosinases in hydrolyzing anserine using a sensitive LC-MS/MS approach.

Crystal structure of aspartyl dipeptidase from Xenopus laevis revealed ligand binding induced loop ordering and catalytic triad assembly.

Gene encoding aspartyl dipeptidase from Xenopus levies (PepExl) is upregulated by thyroid hormone and is proposed to play a significant role in resorption of tadpole tail during metamorphosis. However, the importance of peptidase activity for the resorption of the tail remain elusive. Here we report the crystal structures of first eukaryotic S51 peptidase, PepExl, in its ligand-free and Asp-bound states at 1.4 and 1.8 Å resolutions, respectively. The active site is located at dimeric interface and the catalytic triad is found to be dissembled in ligand-free and assembled in Asp-bound state. Structural comparison and molecular dynamic simulations of ligand-free and Asp-bound states shows that distinct loop (loop-A) plays an important role in active site shielding, substrate binding and enzyme activation. This study illuminates the Asp-X dipeptide binding in PepExl is associated with ordering of the loop-A and assembly of residues of catalytic triad in active conformation for enzymatic activity.

Co-expression with chaperones can affect protein 3D structure as exemplified by loss-of-function variants of human prolidase.

Prolidase catalyzes the cleavage of dipeptides containing proline on their C terminus. The reduction in prolidase activity is the cause of a rare disease named 'Prolidase Deficiency'. Local structural disorder was indicated as one of the causes for diminished prolidase activity. Previous studies showed that heat shock proteins can partially recover prolidase activity in vivo. To analyze this mechanism of enzymatic activity rescue, we compared the crystal structures of selected prolidase mutants expressed in the absence and in the presence of chaperones. Our results confirm that protein chaperones facilitate the formation of more ordered structures by their substrate protein. These results also suggest that the protein expression system needs to be considered as an important parameter in structural studies. DATABASES: The reported crystal structures and their associated structure factor amplitudes were deposited in the Protein Data Bank under the accession codes 6SRE, 6SRF, and 6SRG, respectively.

Structure of human DPEP3 in complex with the SC-003 antibody Fab fragment reveals basis for lack of dipeptidase activity.

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.

Engineering Xaa-Pro dipeptidyl aminopeptidase for specific cleavage of glucagon and glucagon-like peptide 1 from fusion proteins.

N-terminal extensions ("tags") have proven valuable for producing peptides using high throughput recombinant expression technologies. However, the applicability is hampered by the limited options for specific and efficient proteases to release the fully native sequence without additional amino acids in the N-terminal. Here we describe the Escherichia coli (E. coli) expression, purification and characterization of engineered variants of Xaa-Pro dipeptidyl aminopeptidase (Xaa-Pro-DAP) derived from Lactococcus lactis for cleavage of Gly-Pro dipeptide extension in the N-terminal of glucagon and glucagon-like peptide 1 (GLP-1(7-37)). By single amino acid substitution in the Xaa-Pro-DAP protease, significantly higher product yields were achieved. The combination of HRV14 3C protease and engineered Xaa-Pro-DAP is suggested for obtaining native N-terminal of peptides.

Characterization of substrate specificity and novel autoprocessing mechanism of dipeptidase A from Prevotella intermedia.

Prevotella intermedia, a Gram-negative anaerobic rod, is frequently observed in subgingival polymicrobial biofilms from adults with chronic periodontitis. Peptidases in periodontopathic bacteria are considered to function as etiological reagents. Prevotella intermedia OMA14 cells abundantly express an unidentified cysteine peptidase specific for Arg-4-methycoumaryl-7-amide (MCA). BAU17746 (locus tag, PIOMA14_I_1238) and BAU18827 (locus tag, PIOMA14_II_0322) emerged as candidates of this peptidase from the substrate specificity and sequence similarity with C69-family Streptococcus gordonii Arg-aminopeptidase. The recombinant form of the former solely exhibited hydrolyzing activity toward Arg-MCA, and BAU17746 possesses a 26.6% amino acid identity with the C69-family Lactobacillus helveticus dipeptidase A. It was found that BAU17746 as well as L. helveticus dipeptidase A was a P1-position Arg-specific dipeptidase A, although the L. helveticus entity, a representative of the C69 family, had been reported to be specific for Leu and Phe. The full-length form of BAU17746 was intramolecularly processed to a mature form carrying the N-terminus of Cys15. In conclusion, the marked Arg-MCA-hydrolyzing activity in Pre. intermedia was mediated by BAU17746 belonging to the C69-family dipeptidase A, in which the mature form carries an essential cysteine at the N-terminus.

Affinity Immobilization of a Bacterial Prolidase onto Metal-Ion-Chelated Magnetic Nanoparticles for the Hydrolysis of Organophosphorus Compounds.

In this study, silica-coated magnetic nanoparticles (SiMNPs) with isocyanatopropyltriethoxysilane as a metal-chelating ligand were prepared for the immobilization of His6-tagged Escherichia coli prolidase (His6-EcPepQ). Under one-hour coupling, the enzyme-loading capacity for the Ni2+-functionalized SiMNPs (NiNTASiMNPs) was 1.5 mg/mg support, corresponding to about 58.6% recovery of the initial activity. Native and enzyme-bound NiNTASiMNPs were subsequently characterized by transmission electron microscopy (TEM), superparamagnetic analysis, X-ray diffraction, and Fourier transform infrared (FTIR) spectroscopy. As compared to free enzyme, His6-EcPepQ@NiNTASiMNPs had significantly higher activity at 70 °C and pH ranges of 5.5 to 10, and exhibited a greater stability during a storage period of 60 days and could be recycled 20 times with approximately 80% retention of the initial activity. The immobilized enzyme was further applied in the hydrolysis of two different organophosphorus compounds, dimethyl p-nitrophenyl phosphate (methyl paraoxon) and diethyl p-nitrophenyl phosphate (ethyl paraoxon). The experimental results showed that methyl paraoxon was a preferred substrate for His6-EcPepQ and the kinetic behavior of free and immobilized enzymes towards this substance was obviously different. Taken together, the immobilization strategy surely provides an efficient means to deposit active enzymes onto NiNTASiMNPs for His6-EcPepQ-mediated biocatalysis.

Quantitation of Methionine Sulfoxide in Milk and Milk-Based Beverages-Minimizing Artificial Oxidation by Anaerobic Enzymatic Hydrolysis.

Protein oxidation in milk products may entail flavor changes through reactions at methionine residues. However, little is known about the extent of methionine oxidation in milk and milk products. In the present study, a method for quantitation of methionine, methionine sulfoxide, and methionine sulfone by a stable isotope dilution assay using HILIC-ESI-MS/MS was established. For the quantitation of protein-bound analytes, anaerobic enzymatic hydrolysis was optimized to suppress artificial methionine oxidation. Moreover, the method allowed for monitoring of artificial oxidation by coincubation of the labeled probe [2H8]methionine. The percentage of oxidized methionine was low in UHT milk (up to 1.6%) and evaporated milk (up to 8.8%), but higher in beverages such as cocoa milk drinks (up to 19.0%) and coffee milk drinks (up to 32.8%), resulting in methionine sulfoxide concentrations of up to 6.7 g/kg protein in the latter. These products are important dietary sources of methionine sulfoxide. Model studies revealed that methionine residues can be oxidized strongly in the presence of phenolic compounds such as catechin, caffeic acid, and gallic acid, which are present in cocoa and coffee and may account for the high extent of oxidation in commercial samples.

Focusing on the functional characterization of the anserinase from Oreochromis niloticus.

Carnosine, anserine and homocarnosine are the three most representative compounds of the histidine dipeptides family, widely distributed in mammals in different amounts depending on the species and the tissue considered. Histidine dipeptides are mainly degraded by two different carnosinase homologues: a highly specific metal-ion dependent carnosinase (CN1) located in serum and brain and a non-specific cytosolic form (CN2). The hydrolysis of such dipeptides in prokaryotes and eukaryotes is also catalyzed by the anserinase (ANSN). Such naturally occurring dipeptides represent an interesting topic because they seem to have numerous biological roles such as potential neuroprotective and neurotransmitter functions in the brain and therefore ANSN results to be a very interesting target of study. We here report, for the first time, cloning, expression of ANSN from the fish Oreochromis niloticus both in a mammalian and in a prokaryotic system, in order to perform deep functional studies by enzymatic assays in the presence of different metals and substrates. Furthermore, by means of a mass spectrometry-based proteomic approach, we analysed protein sequence and the potential presence of post-translational modifications in the mammalian recombinant protein. Finally, a preliminary structural characterization was carried out on ANSN produced in Escherichia coli.

Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases.

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.

Development of Low-Molecular Weight Collagen Peptide Complex with Glycosaminoglycan Components.

Enzymatic hydrolysis of biopolymers of the cartilage tissue was studied for obtaining a complex of type II collagen peptides and glycosaminoglycan oligosaccharides. Hydrothermal hydrolysis in a high pressure homogenizer followed by enzymatic hydrolysis of the cartilage tissue biopolymers with proteolytic enzyme preparation Karipazim yielded a complex of collagen peptides and glycosaminoglycan oligosaccharides with molecular weights of 240-720 Da. Low molecular weight of the components increases their bioavailability. Entering into the cells (chondrocytes), low-molecular-weight peptides, disaccharides, and oligosaccharides as structural elements of the matrix can participate in the formation of fibrils of collagen and proteoglycans. Exogenous substances replenish deficient components of the matrix and/or their concentrations, affect the formation and strengthen the cartilage tissue. Thus, using cattle and porcine hyaline cartilages, we prepared a complex of biopolymers with lower molecular weights in comparison with previously developed nutraceuticals.

Structural basis for prolidase deficiency disease mechanisms.

Prolidase is a metallopeptidase that cleaves iminodipeptides containing a proline (Pro) or hydroxyproline (Hyp) residue at their C-terminal end. The disease prolidase deficiency (PD) is a rare recessive human disorder characterized by reduced prolidase activity. PD manifests itself by a wide range of severe clinical symptoms, most commonly as skin ulceration, recurrent infections of the respiratory tract, and mental retardation. Several mutations in the PEPD gene have been identified that are responsible for the loss or the reduction of prolidase activity. In contrast, the structural basis of enzyme inactivation has so far remained elusive. In this study, we present high resolution crystal structures of a number of human prolidase (HsProl) variants, in which single amino acids are either substituted by others or deleted. The observed implications of the mutations on the three-dimensional structure of HsProl are reported and discussed and related to their enzymatic activity. The resulting structures may be divided into four groups depending on the presumed effect of the corresponding mutations on the reaction mechanism. The four possible inactivation mechanisms, which could be elucidated, are disruption of the catalytic Mn2 (OH- )-center, introduction of chain disorder along with the displacement of important active site residues, rigidification of the active site, and flexibilization of the active site. DATABASE: All refined structure coordinates as well as the corresponding structure factor amplitudes have been deposited in the PDB under the accession numbers 5MBY, 5MBZ, 5MC0, 5MC1, 5MC2, 5MC3, 5MC4, 5MC5, 6H2P, 6H2Q.

Comprehensive in-silico prediction of damage associated SNPs in Human Prolidase gene.

Prolidase is cytosolic manganese dependent exopeptidase responsible for the catabolism of imido di and tripeptides. Prolidase levels have been associated with a number of diseases such as bipolar disorder, erectile dysfunction and varied cancers. Single nucleotide polymorphism present in coding region of proteins (nsSNPs) has the potential to alter the primary structure as well as function of the protein. Hence, it becomes necessary to differentiate the potential harmful nsSNPs from the neutral ones. 19 nsSNPs were predicted as damaging by in-silico analysis of 298 nsSNPs retrieved from dbSNP database. Consurf analysis showed 18 out of 19 substitutions were present in the conserved regions. 4 substitutions (D276N, D287N, E412K, and G448R) that observed to have damaging effect are present in catalytic pocket. Four SNPs listed in splice site region were found to affect splicing of mRNA by altering acceptor site. On 3'UTR scan of 77 SNPs listed in SNP database, 9 SNPs were lead to alter miRNA target sites. These results provide a filtered data to explore the effect of uncharacterized nsSNP and SNP related to UTRs and splice site of prolidase to find their association with the disease susceptibility and to design the target dependent drugs for therapeutics.

Trichomonas vaginalis metalloproteinase TvMP50 is a monomeric Aminopeptidase P-like enzyme.

Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric active enzyme. This was an unexpected finding since other Xaa-Pro aminopeptidases/prolidases are active as a biological unit formed by dimers/tetramers. In this study, the evolutionary history of TvMP50 and the preliminary crystal structure of the recombinant enzyme determined at 3.4 Å resolution is reported. TvMP50 was shown to be a type of putative, eukaryotic, monomeric aminopeptidase P, and the crystallographic coordinates showed a monomer on a "pseudo-homodimer" array on the asymmetric unit that resembles the quaternary structure of the M24B dimeric family and suggests a homodimeric aminopeptidase P-like enzyme as a likely ancestor. Interestingly, TvMP50 had a modified N-terminal region compared with other Xaa-Pro aminopeptidases/prolidases with three-dimensional structures; however, the formation of the standard dimer is structurally unstable in aqueous solution, and a comparably reduced number of hydrogen bridges and lack of saline bridges were found between subunits A/B, which could explain why TvMP50 portrays monomeric functionality. Additionally, we found that the Parabasalia group contains two protein lineages with a "pita bread" fold; the ancestral monomeric group 1 was probably derived from an ancestral dimeric aminopeptidase P-type enzyme, and group 2 has a probable dimeric kind of ancestral eukaryotic prolidase lineage. The implications of such hypotheses are also presented.

Structures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer.

Dipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases (preferentially postproline) associated with pathophysiological roles in immune response and cancer biology. While the DPP family member DPP4 is extensively characterized in molecular terms as a validated therapeutic target of type II diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8 and DPP9 have not been reported. In this study we describe crystal and molecular structures of human DPP8 (2.5 Å) and DPP9 (3.0 Å) unliganded and complexed with a noncanonical substrate and a small molecule inhibitor, respectively. Similar to DPP4, DPP8 and DPP9 molecules consist of one ß-propeller and α/ß hydrolase domain, forming a functional homodimer. However, they differ extensively in the ligand binding site structure. In intriguing contrast to DPP4, where liganded and unliganded forms are closely similar, ligand binding to DPP8/9 induces an extensive rearrangement at the active site through a disorder-order transition of a 26-residue loop segment, which partially folds into an α-helix (R-helix), including R160/133, a key residue for substrate binding. As vestiges of this helix are also seen in one of the copies of the unliganded form, conformational selection may contributes to ligand binding. Molecular dynamics simulations support increased flexibility of the R-helix in the unliganded state. Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism. DPP8 and DPP9 are closely similar and display few opportunities for targeted ligand design. However, extensive differences from DPP4 provide multiple cues for specific inhibitor design and development of the DPP family members as therapeutic targets or antitargets.