Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes

D-psicose, a rare sugar produced by the enzymatic reaction of D-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C-3 position, with marked efficiency for D-psicose. The crystal structures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the beta4-alpha4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.     

Characterization of an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to D-psicose

The noncharacterized gene previously proposed as the D-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from D-fructose to D-psicose. The enzyme exhibited maximal activity at 50 degrees C and pH 8.0 with Mn2+. The turnover number (k(cat)) and catalytic efficiency (k(cat)/Km) of the enzyme for D-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not D-tagatose 3-epimerase but D-psicose 3-epimerase. The equilibrium ratio between D-psicose and D-fructose was 32:68 at 30 degrees C. D-Psicose was produced at 230 g/liter from 700-g/liter D-fructose at 50 degrees C after 100 min, corresponding to a conversion yield of 32.9%.     

以D-果糖为原料利用新型异构酶转化生产D-阿洛糖

稀少糖是自然界中含量稀少、化学合成困难的一类低热量单糖。D-阿洛糖是一种重要的稀少己醛糖,其具有减少活性自由基、抑制癌细胞增殖等独特的生理学功能。因此,以微生物发酵生产D-阿洛酮糖-3-差向异构酶(DPE)和L-鼠李糖异构酶(L-RhI)转化生产D-阿洛糖,成为近几年来国际研究的热点之一。文中分别克隆了来源于解纤维梭菌Clostridium cellulolyticum H10的DPE基因以及来源于枯草芽胞杆菌Bacillussubtilis 168的L-RhI基因,并分别使其在宿主菌B.subtilis及大肠杆菌Escherichia coli BL21(DE3)中得到了表达。进一步利用镍亲和层析和阴离子交换色谱等手段对这两种酶进行了纯化,并对这两种纯化后酶的转化能力进行了分析测定。结果表明,以D-果糖为原料利用两种异构酶依次转化获得D-阿洛酮糖及D-阿洛糖,其两步转化效率分别为27.34%和34.64%。     

3D-structure of human estrogenic 17beta-HSD1: binding with various steroids.

Human estrogenic dehydrogenase (17β-HSD1) catalyses the last step in the biosynthesis of the active estrogens that stimulate the proliferation of breast cancer cells. While the primary substrate for the enzyme is estrone, the enzyme has some activity for the non-estrogenic substrates. To better understand the structure–function relationships of 17β-HSD1 and to provide a better ground for the design of inhibitors, we have determined the crystal structures of 17β-HSD1 in complex with different steroids.

The structure of the complex of estradiol with the enzyme determined previously (Azzi et al., Nature Structural Biology 3, 665–668) showed that the narrow active site was highly complementary to the substrate. The substrate specificity is due to a combination of hydrogen bonding and hydrophobic interactions between the steroid and the enzyme binding pocket. We have now determined structures of 17β-HSD1 in complex with dihydrotestosterone and 20-OH-progesterone. In the case of the C19 androgen, several residues within the enzyme active site make some small adjustments to accommodate the increased bulk of the substrate. In addition, the C19 steroids bind in a slightly different position from estradiol with shifts in positions of up to 1.4 Å. The altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished). The known kinetic parameters for these substrates can be rationalized in light of the structures presented. These results give evidence for the structural basis of steroid recognition by 17β-HSD1 and throw light on the design of new inhibitors for this pivotal steroid enzyme.     

Crystal structures of substrate free and complex forms of reactivated BphC, an extradiol type ring-cleavage dioxygenase

BphC derived from Pseudomonas sp. strain KKS102, an extradiol type catecholic dioxygenase, is a non-heam iron-containing enzyme, playing an important role in the degradation of biphenyl/PCB (Poly Chlorinated Biphenyls) in the microbe. Although we had earlier solved the crystal structure of KKS102 BphC, it was the inactive form with Fe(III) in the active site. In order to determine the active form structure, BphC was re-activated by anaerobic incubation with Fe(II) and ascorbate, and crystallized anaerobically. The crystal structures of activated BphC and its substrate complex (E x S complex) were determined at 2.0 A resolution under cryogenic condition. In addition, crystal structures of unactivated BphC in substrate free and complex forms were also re-determined. Comparison of activated and unactivated E x S complexes reveals that the orientation of the bound substrate in the active site is significantly different between the two. The structural comparison of the substrate free and complex forms of activated BphC show certain small conformational shifts around the active site upon substrate binding. As a result of the conformational shifts, His194, which has been suggested as the catalytic base, takes part in a weak hydrogen bond with hydroxyl group of the substrate.     

X-ray structures of the <Emphasis Type="Italic">Pseudomonas cichorii</Emphasis> D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates

Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.     

Recent advances on applications and biotechnological production of d-psicose

D-Psicose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of D-fructose and is rarely found in nature. It has 70 % relative sweetness but 0.3 % energy of sucrose, and is suggested as an ideal sucrose substitute for food products. It shows important physiological functions, such as blood glucose suppressive effect, reactive oxygen species scavenging activity, and neuroprotective effect. It also improves the gelling behavior and produces good flavor during food process. This article presents a review of recent studies on the properties, physiological functions, and food application of D-psicose. In addition, the biochemical properties of D-tagatose 3-epimerase family enzymes and the D-psicose-producing enzyme are compared, and the biotechnological production of D-psicose from D-fructose is reviewed.     

Structure analysis of the extracellular domain reveals disulfide bond forming-protein properties of Mycobacterium tuberculosis Rv2969c

Disulfide bond-forming (Dsb) protein is a bacterial periplasmic protein that is essential for the correct folding and disulfide bond formation of secreted or cell wallassociated proteins. DsbA introduces disulfide bonds into folding proteins, and is re-oxidized through interaction with its redox partner DsbB. Mycobacterium tuberculosis, a Gram-positive bacterium, expresses a DsbA-like protein ( Rv2969c), an extracellular protein that has its Nterminus anchored in the cell membrane. Since Rv2969c is an essential gene, crucial for disulfide bond formation, research of DsbA may provide a target of a new class of anti-bacterial drugs for treatment of M.tuberculosis infection. In the present work, the crystal structures of theextracellular region of Rv2969c (Mtb DsbA) were determined in both its reduced and oxidized states. The overall structure of Mtb DsbA can be divided into two domains: a classical thioredoxin-like domain with a typical CXXC active site, and an α-helical domain. It largely resembles its Escherichiacoli homologue EcDsbA, however, it possesses a truncated binding groove; in addition, its active site is surrounded by an acidic, rather than hydrophobic surface. In our oxidoreductase activity assay, Mtb DsbA exhibited a different substrate specificity when compared to EcDsbA. Moreover, structural analysis revealed a second disulfide bond in Mtb DsbA, which is rare in the previously reported DsbA structures, and is assumed to contribute to the overall stability of Mtb DsbA. To investigate the disulphide formation pathway in M.tuberculosis, we modeled Mtb Vitamin K epoxide reductase (Mtb VKOR), a binding partner of Mtb DsbA, to Mtb DsbA.     

The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The sugar phosphate specificity of the active site of 6-phosphofructo-2-kinase and of the inhibitory site of fructose-2,6-bisphosphatase was investigated. The Michaelis constants and relative Vmax values of the sugar phosphates for the 6-phosphofructo-2-kinase were: D-fructose 6-phosphate, Km = 0.035 mM, Vmax = 1; L-sorbose 6-phosphate, Km = 0.175 mM, Vmax = 1.1; D-tagatose 6-phosphate, Km = 15 mM, Vmax = 0.15; and D-psicose 6-phosphate, Km = 7.4 mM, Vmax = 0.42. The enzyme did not catalyze the phosphorylation of 1-O-methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, 2,5-anhydro-D-mannitol 6-phosphate, D-ribose 5-phosphate, or D-arabinose 5-phosphate. These results indicate that the hydroxyl group at C-3 of the tetrahydrofuran ring must be cis to the beta-anomeric hydroxyl group and that the hydroxyl group at C-4 must be trans. The presence of a hydroxymethyl group at C-2 is required; however, the orientation of the phosphonoxymethyl group at C-5 has little effect on activity. Of all the sugar monophosphates tested, only 2,5-anhydro-D-mannitol 6-phosphate was an effective inhibitor of the kinase with a Ki = 95 microM. The sugar phosphate specificity for the inhibition of the fructose-2,6-bisphosphatase was similar to the substrate specificity for the kinase. The apparent I0.5 values for inhibition were: D-fructose 6-phosphate, 0.01 mM; L-sorbose 6-phosphate, 0.05 mM; D-psicose 6-phosphate, 1 mM; D-tagatose 6-phosphate, greater than 2 mM; 2,5-anhydro-D-mannitol 6-phosphate, 0.5 mM. 1-O-Methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, and D-arabinose 5-phosphate did not inhibit. Treatment of the enzyme with iodoacetamide decreased sugar phosphate affinity in the kinase reaction but had no effect on the sensitivity of fructose-2,6-bisphosphatase to sugar phosphate inhibition. The results suggest a high degree of homology between two separate sugar phosphate binding sites for the bifunctional enzyme.     

The putative propeptide of MycP1 in mycobacterial type VII secretion system does not inhibit protease activity but improves protein stability

Mycosin-1 protease (MycP1) is a serine protease anchored to the inner membrane of Mycobacterium tuberculosis, and is essential in virulence factor secretion through the ESX-1 type VII secretion system (T7SS). Bacterial physiology studies demonstrated that MycP1 plays a dual role in the regulation of ESX-1 secretion and virulence, primarily through cleavage of its secretion substrate EspB. MycP1 contains a putative N-terminal inhibitory propeptide and a catalytic triad of Asp-His-Ser, classic hallmarks of a subtilase family serine protease. The MycP1 propeptide was previously reported to be initially inactive and activated after prolonged incubation. In this study, we have determined crystal structures of MycP1 with (MycP1^24-422) and without (MycP1^63-422) the propeptide, and conducted EspB cleavage assays using the two proteins. Very high structural similarity was observed in the two crystal structures. Interestingly, protease assays demonstrated positive EspB cleavage for both proteins, indicating that the putative propeptide does not inhibit protease activity. Molecular dynamic simulations showed higher rigidity in regions guarding the entrance to the catalytic site in MycP1^24-422 than in MycP1^63-422, suggesting that the putative propeptide might contribute to the conformational stability of the active site cleft and surrounding regions.     

Effects of dietary D-psicose on diurnal variation in plasma glucose and insulin concentrations of rats

The effects of supplemental D-psicose in the diet on diurnal variation in plasma glucose and insulin concentrations were investigated in rats. Forty-eight male Wistar rats were divided into four groups. Each group except for the control group was fed a diet of 5% D-fructose, D-psicose, or psico-rare sugar (3:1 mixture of D-fructose and D-psicose) for 8 weeks. Plasma glucose levels were lower and plasma insulin levels were higher at all times of day in the psicose and psico-rare sugar groups than in the control and fructose groups. Weight gain was significantly lower in the psicose group than in the control and fructose groups. Liver glycogen content, both before and after meals was higher in the psicose group than in the control and fructose groups. These results suggest that supplemental D-psicose can lower plasma glucose levels and reduce body fat accumulation. Hence, D-psicose might be useful in preventing postprandial hyperglycemia in diabetic patients.     

Crystal structures of E. coli nicotinate mononucleotide adenylyltransferase and its complex with deamido-NAD.

Nicotinamide/Nicotinate mononucleotide (NMN/NaMN) adenylyltransferase is an indispensable enzyme in both de novo biosynthesis and salvage of NAD+ and NADP+. In prokaryotes, it is absolutely required for cell survival, thus representing an attractive target for the development of new broad-spectrum antibacteria inhibitors. The crystal structures of E. coli NaMN adenylyltransferase (NMNAT) and its complex with deamido-NAD (NaAD) revealed that ligand binding causes large conformational changes in several loop regions around the active site. The enzyme specifically recognizes the deamidated pyridine nucleotide through interactions between nicotinate carboxylate with several protein main chain amides and a positive helix dipole. Comparison of E. coli NMNAT with those from archaeal organisms revealed extensive differences in the active site architecture, enzyme-ligand interaction mode, and bound dinucleotide conformations. The bacterial NaMN adenylyltransferase structures described here provide a foundation for structure-based design of specific inhibitors that may have therapeutic potential.     

The enzymatic activity of Arabidopsis protein arginine methyltransferase 10 is essential for flowering time regulation

Arabidopsis AtPRMT10 is a plant-specific type I protein arginine methyltransferase that can asymmetrically dimethylate arginine 3 of histone H4 with auto-methylation activity. Mutations of AtPRMT10 derepress FLOWERING LOCUS C (FLC) expression resulting in a late-flowering phenotype. Here, to further investigate the biochemical characteristics of AtPRMT10, we analyzed a series of mutated forms of the AtPRMT10 protein. We demonstrate that the conserved “VLD” residues and “double-E loop” are essential for enzymatic activity of AtPRMT10. In addition, we show that Arg54 and Cys259 of AtPRMT10, two residues unreported in animals, are also important for its enzymatic activity. We find that Arg13 of AtPRMT10 is the auto-methylation site. However, substitution of Arg13 to Lys13 does not affect its enzymatic activity. In vivo complementation assays reveal that plants expressing AtPRMT10 with VLD-AAA, E143Q or E152Q mutations retain high levels of FLC expression and fail to rescue the late-flowering phenotype of atprmt10 plants. Taken together, we conclude that the methyltransferase activity of AtPRMT10 is essential for repressing FLC expression and promoting flowering in Arabidopsis.     

新型低热量甜味剂——D-阿洛酮糖的研究进展

寻找适合糖尿病人服用的甜味剂具有重要的经济价值。D-阿洛酮糖是D-果糖的C3差向异构体,是一种稀有糖。作为一种新型低热量甜味剂,D-阿洛酮糖近年来日益引起人们的重视。本文对国际上近年来发表的关于D-阿洛酮糖的药理活性资料进行了系统的总结。D-阿洛酮糖几乎不提供热量,具有显著的降血糖、降血脂效果,对糖尿病动物的胰岛β细胞有明显的保护作用。此外,D-阿洛酮糖无毒副作用,是一种食用安全的糖。这些研究结果说明:D-阿洛酮糖在预防和治疗糖尿病方面具有极大的潜力,可以作为糖尿病人食用的新型甜味剂,具有良好的市场前景。     

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.     

Structure of the catalytic domain of a state transition kinase homolog from Micromonas algae

Under natural environments, plants and algae have evolved various photosynthetic acclimation mechanisms in response to the constantly changing light conditions. The state transition and long-term response processes in photosynthetic acclimation involve remodeling and composition alteration of thylakoid membrane. A chloroplast protein kinase named Stt7/STN7 has been found to have pivotal roles in both state transition and longterm response. Here we report the crystal structures of the kinase domain of a putative Stt7/STN7 homolog from Micromonas sp. RCC299 (MsStt7d) in the apo form and in complex with various nucleotide substrates. MsStt7d adopts a canonical protein kinase fold and contains all the essential residues at the active site. A novel hairpin motif, found to be a conserved feature of the Stt7/STN7 family and indispensable for the kinase stability, interacts with the activation loop and fi xes it in an active conformation. We have also demonstrated that MsStt7d is a dualspecifi city kinase that phosphorylates both Thr and Tyr residues. Moreover, preliminary in vitro data suggest that it might be capable of phosphorylating a consensus N-terminal pentapeptide of light-harvesting proteins Micromonas Lhcp4 and Arabidopsis Lhcb1 directly. The potential peptide/protein substrate binding site is predicted based on the location of a pseudo-substrate contributed by the adjacent molecule within the crystallographic dimer. The structural and biochemical data presented here provide a framework for an improved understanding on the role of Stt7/STN7 in photosynthetic acclimation.     

The crystal structure and mechanism of 1-L-myo-inositol- 1-phosphate synthase.

1-l-myo-Inositol-1-phosphate synthase catalyzes the conversion of d-glucose 6-phosphate to 1-l-myo-inositol-1-phosphate (MIP), the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, intramolecular aldol cyclization, and reduction. We have determined the first crystal structure of MIP synthase. We present structures of both the NAD-bound enzyme and the enzyme bound to an inhibitor, 2-deoxy-glucitol-6-phosphate. While 58 amino acids are disordered in the unbound form of the enzyme in the vicinity of the active site, the inhibitor nucleates the folding of this domain in a striking example of induced fit, serving to completely encapsulate it within the enzyme. Three helices and a long beta-strand are formed in this process. We postulate a mechanism for the conversion based on the structure of the inhibitor-bound complex.     

Mechanism of the Schiff base forming fructose-1,6-bisphosphate aldolase: structural analysis of reaction intermediates

The glycolytic enzyme fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis of Schiff base forming class I FBPA relies on a number of intermediates covalently bound to the catalytic lysine. Using active site mutants of FBPA I from Thermoproteus tenax, we have solved the crystal structures of the enzyme covalently bound to the carbinolamine of the substrate fructose 1,6-bisphosphate and noncovalently bound to the cyclic form of the substrate. The structures, determined at a resolution of 1.9 A and refined to crystallographic R factors of 0.148 and 0.149, respectively, represent the first view of any FBPA I in these two stages of the reaction pathway and allow detailed analysis of the roles of active site residues in catalysis. The active site geometry of the Tyr146Phe FBPA variant with the carbinolamine intermediate supports the notion that in the archaeal FBPA I Tyr146 is the proton donor catalyzing the conversion between the carbinolamine and Schiff base. Our structural analysis furthermore indicates that Glu187 is the proton donor in the eukaryotic FBPA I, whereas an aspartic acid, conserved in all FBPA I enzymes, is in a perfect position to be the general base facilitating carbon-carbon cleavage. The crystal structure of the Trp144Glu, Tyr146Phe double-mutant substrate complex represents the first example where the cyclic form of beta-fructose 1,6-bisphosphate is noncovalently bound to FBPA I. The structure thus allows for the first time the catalytic mechanism of ring opening to be unraveled.     

Tobacco etch virus proteinase: crystal structure of the active enzyme and its inactive mutant

Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 A resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217-221 of the enzyme are involved in formation of the binding pockets S3-S6. This indicates that the autolysis of the peptide bond Met218-Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in considerable decrease in the enzymatic activity.