The role of Arg114 at subsites E and F in reactions catalyzed by hen egg-white lysozyme

To understand better the role of subsites E and F in lysozyme-catalyzed reactions, mutant enzymes, in which Arg114, located on the right side of subsites E and F in hen egg-white lysozyme (HEL), was replaced with Lys, His, or Ala, were prepared. Replacement of Arg114 with His or Ala decreased hydrolytic activity toward an artificial substrate, glycol chitin, while replacement with Lys had little effect. Kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed that the replacement for the Arg residue reduced the binding free energies of E-F sites and the rate constant of transglycosylation. The rate constant of transglycosylation for R114A was about half of that for the wild-type enzyme. (1)H-NMR analysis of R114H and R114A indicated that the structural changes induced by the mutations were not restricted to the region surrounding Arg114, but rather extended to the aromatic side chains of Phe34 and Trp123, of which the signals are connected with each other through nuclear Overhauser effect (NOE) in the wild-type. We speculate that such a conformational change causes differences in substrate and acceptor binding at subsites E and F, lowering the efficiency of glycosyl transfer reaction of lysozyme.     

Enzymatic properties of rhea lysozyme

Rhea lysozyme was analyzed for its enzymatic properties both lytic and oligomer activities to reveal the structural and functional relationships of goose type lysozyme. Rhea lysozyme had the highest lytic activity at pH 6, followed by ostrich and goose at pH 5.5-6, whereas the optimum of cassowary was at pH 5. pH profile was correlated to the net charge of each molecule surface. On the other hand, the pH optimum for oligomer substrate was found to be pH 4, indicating the mechanism of rhea catalysis as a general acid. The time-course of the reaction was studied using beta-1,4-linked oligosaccharide of N-acetylglucosamine (GlcNAc) with a polymerization degree of n ((GlcNAc)n) (n=4, 5, and 6) as the substrate. This enzyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3+(GlcNAc)3 predominating over that to (GlcNAc)2+ (GlcNAc)4. This indicates that the lysozyme hydrolyzed preferentially the third glycosidic linkage from the nonreducing end. Theoretical analysis has shown the highest rate constant value at 1.5 s-1 with (GlcNAc)6. This confirmed six substrate binding subsites as goose lysozyme (Honda, Y., and Fukamizo, T., Biochim. Biophys. Acta, 1388, 53-65 (1998)). The different binding free energy values for subsites B, C, F, and G from goose lysozyme might responsible for the amino acid substitutions, Asn122Ser and Phe123Met, located at the subsite B.     

Histidine-114 at subsites E and F can explain the characteristic enzymatic activity of guinea hen egg-white lysozyme

The courses of the reaction catalyzed by guinea hen egg-white lysozyme (GHL), in which Asn113 and Arg114 at subsites E and F in hen egg-white lysozyme (HEL) are replaced by Lys and His, respectively, was studied with the substrate N-acetylglucosamine pentamer, (GlcNAc)5. Although GHL was found to retain the main-chain folding similar to HEL as judged from CD spectroscopy, the courses of GHL showed increased production of (GlcNAc)4 and reduced production of (GlcNAc)2 when compared with HEL. To identify critical residue(s) involved in the alteration in the courses of GHL, two mutant enzymes as to subsites E and F in HEL, N113K and R114H, were prepared by site-directed mutagenesis. Kinetic analysis of these mutants revealed that the mutation of Asn113 to Lys had little effect on the courses of HEL, while the Arg114 to His mutation completely reproduced the courses of GHL, demonstrating that His114 in GHL is the key residue responsible for the characteristic courses of GHL. Computer simulation of the reaction courses of the R114H mutant revealed that this substitution decreased not only the binding free energies for subsites E and F, but also the rate constant of transglycosylation. The Arg residue at position 114 may play an important role in the transglycosylation activity of HEL.     

Functional and structural effects of mutagenic replacement of Asn37 at subsite F on the lysozyme-catalyzed reaction

To investigate the functional role of subsites E and F in lysozyme catalysis, Asn37 of hen egg-white lysozyme (HEL), which is postulated to participate in sugar residue binding at the right-sided subsite F through hydrogen bonding, was replaced by Ser or Gly by site-directed mutagenesis. The mutations of Asn37 neither significantly affected the binding constant for chitotriose nor the enzymatic activity toward the substrate glycol chitin. However, kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed that the conversion of Asn37 to Gly decreased the binding free energies for subsites E and F, while the conversion to Ser increased the substrate affinity at subsite F. It was further found that the rate constant of transglycosylation was reduced by these mutations. These results suggest that Asn37 is involved not only in substrate binding at subsite F but also in transglycosylation activity. No remarkable change in the tertiary structure except the side chain of the 37th residue was detected on X-ray analysis of the mutant proteins, indicating that the alterations in the enzymatic function between the wild type and mutant enzymes depend on limited structural change around the substitution site. It is thus speculated that the slight conformational difference in the side chain of position 37 may affect the substrate and acceptor binding at subsites E and F, leading to lower the efficiency of the transglycosylation activities of the mutant proteins.     

The simultaneous binding of lanthanide and N-acetylglucosamine inhibitors to hen egg-white lysozyme in solution by 1H and 13C nuclear magnetic resonance.

Lanthanide ions and the N-acetylglucosamine (GlcNAc) sugars are able to bind simultaneously to hen egg-white lysozyme (EC 3.2.1.17). The present study characterizes the properties of the ternary complexes with lysozyme, which involve up to seven paramagnetic lanthanides and two diamagnetic lanthanides, together with alpha GlcNAc, beta GlcNAc, alpha MeGlcNAc and beta MeGlcNAc. pH titrations and binding titrations of the GlcNAc sugars with lysozyme-La(III) complexes show that the GlcNAc sugars bind to at least two independent sites and that one of them competes with La(III) for binding to lysozyme. Given the known binding site of lanthanides at Asp-52 and Glu-35, the competitive binding site of GlcNAc is identified as subsite E. A simple analysis of the paramagnetic-lanthanide-induced shifts shows that the GlcNAc sugar binds in subsite C, in accordance with crystallographic results [Perkins, Johnson, Machin & Phillips (1979) Biochem. J. 181, 21-36]. This finding was refined by several computer analyses of the lanthanide-induced shifts of 17 proton and carbon resonances of beta MeGlcNAc. Good fits were obtained for all the signals, except for two that were affected by exchange broadening phenomena. No distinction could be made between a fit for a two-position model of Ln(III) binding with axial symmetry to lysozyme, according to the crystallographic result, or a one-position model with axial symmetry where the Ln(III) is positioned mid-way between Asp-52 and Glu-35. Although this work establishes the feasibility of lanthanide shift reagents for study of protein-ligand complexes, further work is required to establish the manner in which lanthanides bind to lysozyme in solution.     

Chitinolytic activities in the gut of Aedes aegypti (Diptera:Culicidae) larvae and their role in digestion of chitin-rich structures

Mosquito larvae are believed to be capable of digesting chitin, an insoluble polysaccharide of N-acetylglucosamine, for their nutritional benefit. Studies based on physiological and biochemical assays were conducted in order to detect the presence of chitinase activities in the gut of the detritus-feeding Aedes aegypti larvae. Larvae placed for 24 h in suspensions of chitin azure were able to digest the ingested chitin. Semi-denaturing PAGE using glycol chitin and two fluorogenic substrate analogues showed the presence of two distinct chitinase activities: an endochitinase that catalyzed the hydrolysis of chitin and an endochitinase that cleaved the short substrates [4MU(GlcNAc)(3)] and [4MU(GlcNAc)(2)] that hydrolyzed the chitobioside [4MU(GlcNAc)(2)]. The endochitinase had an extremely broad pH-activity against glycol chitin and chitin azure, pH ranging from 4.0 to 10.0. When the substrate [4MU(GlcNAc)(3)] was used, two activities were observed at pH ranges 4.0-6.0 and 8.0-10.0. Chitinase activity against [4MU(GlcNAc)(3)] was detected throughout the gut with the highest specific activity in the hindgut. The pH of the gut contents was determined by observing color changes in gut after feeding the larvae with color indicator dyes. It was observed a correlation between the pH observed in the gut of feeding larvae (pH 10-6.0) and the optimum pH for gut chitinase activities. In this work, we report that gut chitinases may be involved in the digestion of chitin-containing structures and also in the partial degradation of the chitinous peritrophic matrix in the hindgut.     

Interactions on 3-deoxy and 6-deoxy derivatives of N-acetyl-D-glucosamine with hen lysozyme.

The interactions of deoxy derivatives of GlcNAc, 6-deoxy-GlcNAc, and 3-deoxy-GlcNAc with hen egg-white lysozyme [EC 3.2.1.17] were studied at various pH's by measuring the changes in the circular dichroic (CD) band at 295 nm. It was shown that 6-deoxy-GlcNAc and 3-deoxy-GlcNAc bind at subsite C of lysozyme and compete with GlcNAc. The pH dependence of the binding constant of 6-deoxy-GlcNAc was the same as that of GlcNAc. On the other hand, the binding constants of 3-deoxy-GlcNAc were 3--10 times smaller than those of GlcNAc in the pH range from 3 to 9. X-ray crystallographic studies show that O(6) and O(3) of GlcNAc at subsite C are hydrogen-bonded to the indole NH's of Trp 62 and Trp 63, respectively, but the above results indicate that Trp 63, not Trp 62, is important for the interaction of GlcNAc with lysozyme.     

Difference in substrate binding processes between intact and iodine-inactivated lysozyme.

The binding of glycol chitin to intact and iodine-inactivated lysozyme was studied by measuring the absorbance of the complex with N-methylnicotinamide chloride, which binds to the subsite C in lysozyme as a competitive inhibitor. The association constant of glycol chitin to inactivated lysozyme was determined from static experiments to be 1.7 × 10⁴M^?1. The kinetics of the substrate binding to intact and iodine-inactivated lysozyme were measured by the stopped-flow method at 23°C and pH 5.6. The binding to inactivated lysozyme was clearly monophasic, whereas in intact lysozyme it consisted of multiple phases. In the substrate binding to intact lysozyme, a fast bimolecular process and two subsequent slow unimolecular processes were observed besides the hydrolysis process of polymer substrate. These slow phases were missing completely in inactivated lysozyme. It results from the alteration in the local structure occurring at the subsite D in inactivated lysozyme. These results mean that the slow phases are important for catalytic action of lysozyme. The rate constants of association and dissociation in the fast bimolecular process were determined in this paper. Furthermore, the association constant of the substrate to intact lysozyme was also determined kinetically to be 6.5 × 10³M^?1.     

The amino acid sequence of satyr tragopan lysozyme and its activity

The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temminck's tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcal/mol at subsite A and 0.2 kcal/mol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temminck's tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).     

Crystallographic study of turkey egg-white lysozyme and its complex with a disaccharide

The crystal structure of turkey egg-white lysozyme, determined by the molecular replacement method at 5 Å resolution (Bott & Sarma, 1976) has now been refined to 2.8 Å resolution and a model has been built to fit the electron density. A comparison of the co-ordinates with those of hen lysozyme indicate a rootmean-square deviation of 1.6 Å for all the main-chain and side-chain atoms. A significant difference is observed in the region of residues 98 to 115 of the structure. The molecules are packed in this crystal form with the entire length of the active cleft positioned in the vicinity of the crystallographic 6-fold axis and is not blocked by neighboring molecules. A difference electron density map calculated between crystals of turkey lysozyme soaked in a disaccharide of N-acetyl glucosamine—N-acetyl muramic acid and the native crystals showed a strong positive peak at subsite C, a weak positive peak at subsite D and two strong peaks that correspond to the subsite E and a new subsite F′. This new site F′ is different from the subsite F predicted for the sixth saccharide from model building in hen lysozyme. The interactions between the saccharides bound at subsites E and F′ and the enzyme molecules are discussed.     

Preparation of bioactive and surface functional oligomannosyl neoglycoprotein using extracellular pH-sensitive glycosylation of mutant lysozyme having N-linked signal sequence in yeast

Bioactive oligomannosyl lysozyme with improved surface functionalities was successfully prepared by using an extracellular pH-sensitive glycosylation system for heterogeneous protein in yeast cell. A recombinant Saccharomyces cerevisiae carrying a mutant lysozyme gene encoding the signal sequence of an N-linked glycosylation site at position 49 was cultivated in various pH conditions to investigate the effects of extracellular pH on the glycosylation patterns and the expression of the protein. A large polymannose (Man(310)GlcNAc(2)) chain-linked lysozyme was predominantly expressed accompanied by small amounts of a core-type oligomannose chain (Man(14)GlcNAc(2))-linked lysozyme in the yeast medium where the extracellular pH was kept at 3.5 or above, while an oligomannose chain lysozyme was preferentially expressed in the yeast medium where the pH was less than 3. The lytic activities of the oligomannosyl and the polymannosyl lysozymes were found to be 70.4 and 5.1%, respectively, of the wild-type lysozyme when Micrococcus lysodeikticus cells were used as the substrate. The enzymatic activity of the oligomannosyl lysozyme was totally conserved for the glycolysis assay with a soluble substrate, glycol chitin, whereas that of the polymannosyl lysozyme was not. After heating the sample up to 95 degrees C at pH 7.0 where no visible protein coagulation was observed, thermostability of the enzymatic activity of the oligomannosyl lysozyme was drastically improved with more than 60% of residual lytic activity. Emulsifying properties of the protein also were highly improved by the oligomannosylation, in which the emulsifying activity was 3.2 times higher than that of the wild-type protein. Corresponding to the increase of the surface functionalities, the surface tension of the oligomannosyl protein exhibited a significantly (p < 0.05) lower value compared to that of the wild-type. By using the lower pH medium at 3.0, it was revealed that a substantial amount (0.31 mg/L) of the oligomannosyl lysozyme was successfully obtained in the culture medium. Therefore, the extracellular pH-sensitive glycosylation system can be used to obtain bioactive and surface functional neoglycoproteins.     

The Amino Acid Sequence of Monal Pheasant Lysozyme and Its Activity

The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).     

A novel goose-type lysozyme gene with chitinolytic activity from the moderately thermophilic bacterium <Emphasis Type="Italic">Ralstonia</Emphasis> sp. A-471: cloning,sequencing, and expression

In this study, we cloned the gene encoding goose-type (G-type) lysozyme with chitinase (Ra-ChiC) activity from Ralstonia sp. A-471 genomic DNA library. This is the first report of another type of chitinase after the previously reported chitinases ChiA (Ra-ChiA) and ChiB (Ra-ChiB) in the chitinase system of the moderately thermophilic bacterium, Ralstonia sp. A-471 and also the first such data in Ralstonia sp. G-type lysozyme gene. It consisted of 753 bp nucleotides, which encodes 251 amino acids including a putative signal peptide. This ORF was modular enzyme composed of a signal sequence, chitin-binding domain, linker, and catalytic domain. The catalytic domain of Ra-ChiC showed homologies to those of G-type lysozyme (glycoside hydrolases (GH) family 23, 16.8%) and lysozyme-like enzyme from Clostridium beijerincki (76.1%). Ra-ChiC had activities against ethylene glycol chitin, carboxyl methyl chitin, and soluble chitin but not against the cell wall of Micrococcus lysodeikticus. The enzyme produced α-anomer by hydrolyzing β-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the enzyme, the second and third glycosidic linkage from the non-reducing end were split producing (GlcNAc)2 + (GlcNAc)4 and (GlcNAc)3 + (GlcNAc)3 of almost the same concentration in the early stage of the reaction. The G-type lysozyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3 + (GlcNAc)3 predominating over that to (GlcNAc)2 + (GlcNAc)4. Thus, Ra-ChiC was found to be a novel enzyme in its structural and functional properties. The sequence data reported in the present paper have been submitted to the DDBJ, EMBL, and NCBI databases under the accession number AB45458.     

一种来源于兼性嗜碱菌Bacillus pseudofirmus 703的β-N-乙酰氨基葡萄糖苷酶

N-乙酰氨基葡萄糖苷酶作用于肽聚糖或几丁质,从其非还原末端水解产生β-D-N-乙酰氨基葡萄糖单体,该酶在细胞壁代谢过程中起重要作用,在医药和生物技术领域也有广泛的应用。【目的】克隆表达来源于兼性嗜碱菌Bacillus pseudofirmus 703的β-N-乙酰葡糖胺糖苷酶NagZ703,为获得乙酰氨基葡萄糖单体奠定基础。【方法】以B.pseudofirmus703基因组DNA为模板,克隆得到了β-N-乙酰氨基葡萄糖苷酶基因NagZ703,通过构建pET28a-nagZ703表达载体,在大肠杆菌BL21(DE3)中诱导表达NagZ703,利用镍柱纯化得到NagZ703纯蛋白,并对其酶学和生化性质进行分析。【结果】NagZ703与其同源蛋白多序列比对分析结果表明,NagZ703属于糖苷水解酶3家族(GH3),由2个结构域构成,催化活性中心由位于N端结构域的Arg232-His234-Arg318组成,和研究最多的Bacillussubtilis168来源的BsNagZ氨基酸的序列相似性为37%。酶学性质分析表明,以对硝基酚-β-乙酰氨基葡萄糖苷(pNP-β-GlcNAc)为底物,NagZ703的最适反应温度和pH分别为60°C和pH 6.5,比酶活为10.79 U/mg,其Km和Vmax分别为0.276 mmol/L和0.612 mmol/(mg·min)。该酶具有较好的稳定性,在50°C处理30 min,或在pH 6.0–10.5条件下,4°C保存12 h后,仍保留80%以上的酶活力。EDTA不影响该酶的活性,推测其为非金属依赖酶,且Hg2+可完全抑制酶活性。【结论】本研究将兼性嗜碱菌Bacillus pseudofirmus 703来源的β-N-乙酰葡糖胺糖苷酶NagZ703在大肠杆菌中成功表达和纯化,并分析了其酶学性质;NagZ703的最适pH为6.5,没有表现出耐盐嗜碱的特征;NagZ703能水解胶体几丁质产生GlcNAc,为酶解生产GlcNAc提供了一条可行的思路。     

Binding of substrate analogues to subsites D, E, and F of hen egg-white lysozyme.

The pH dependence of the binding of dye, Beibrich Scarlet, to hen egg-white lysozyme[EC 3.2.1.17] was studied at ionic strength 0.3 and 25 degrees by following circular dichroic (CD)bands originating from the bound dye. This binding involved one of the catalytic groups, Glu 35. The effect of the binding of N-acetylglucosamine (GlcNAc), its dimer or trimer on the binding of this dye was also studied at pH 7.5 by measuring changes in the CD bands of the dye bound to lysozyme. It was shown that there are two sites for simultaneous binding of these saccharides in the lysozyme molecule. The stronger binding of the saccharide was noncompetitive and the weaker binding was competitive with dye binding. The binding constants for the stronger binding site (the upper portion of lysozyme cleft) were in good agreement with those previously determined by following changes in the tryptophyl CD bands of lysozyme. The binding constants to the weaker site were about 1.1 x 10(-4), 5 x 10(2), and 5M(-1) for the trimer, dimer, and monomer of GlcNAc, respectively. Assuming that the trimer, dimer, and monomer occupy subsites D, E, and F; E and F; and E, respectively, the unitary free energies of saccharide binding were estimated to be about --1.9, --3.3, and --2.7 kcal/mole for D, E, and F, respectively.     

Binding of substrate analogs to hen egg-white lysozyme with an ester linkage between Glu 35 and Trp 108.

The interactions of the substrate analogs beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which an ester linkage had been formed between Glu 35 and Trp 108 (108 ester lysozyme), were studied by the circular dichroic and fluorescence techniques, and were compared with those for intact lysozyme. The binding constants of beta-methyl-GlcNAc and (GlcNAc)2 to 108 ester lysozyme were essentially the same as those for intact lysozyme in the pH range of 1 to 5. Above pH 5, the binding constants of these saccharides to 108 ester lysozyme did not change with pH, while the binding constants to intact lysozyme decreased. This indicates that Glu 35 (pK 6.0 in intact lysozyme) participates in the binding of these saccharides. The extent and direction of the pK shifts of Asp 52 (pK 3.5), Asp 48 (pK 4.4), and Asp 66 (pK 1.3) observed when beta-methyl-GlcNAc is bound to 108 ester lysozyme were the same as those for intact lysozyme. The participation of Asp 101 and Asp 66 in the binding of (GlcNAc)2 to 108 ester lysozyme was also the same as that for intact lysozyme. These findings indicate that the conformations of subsites B and C are not changed by the formation of the ester linkage. On the other hand, the binding constants of (GlcNAc)3 to 108 ester lysozyme were higher than those for intact lysozyme at all pH values studied. This result is interpreted in terms of an increase in the affinity for a GlcNAc residue of subsite D, which is situated near the esterified Glu 35.     

Amino acid sequence of Egyptian goose egg-white lysozyme and effects of amino acid substitution on the enzymatic activity

The amino acid sequence of Egyptian goose lysozyme (EGL) from egg-white and its enzymatic properties were analyzed. The established sequence had the highest similarity to wood duck lysozyme (WDL) with five amino acid substitutions, and had eighteen substitutions difference from hen egg-white lysozyme (HEL). Tyr34 and Gly37 were found at subsites E and F of the active site when compared with HEL. The experimental time-course characteristics of EGL against the N-acetylglucosamine pentamer substrate, (GlcNAc)(5), revealed higher production of (GlcNAc)(4) and lower production of (GlcNAc)(2) when compared with HEL. The saccharide-binding ability of subsites A-C in EGL was also found to be weaker than in HEL. An analysis of the enzymatic reactions of five mutants in respect of positions 34, 37 and 71 in HEL indicated the time-course characteristics of EGL to be caused by the combination of three substitutions (F34Y, N37G and G71R) between HEL and EGL. A computer simulation of the EGL-catalyzed reaction suggested that the time-course characteristics of EGL resulted from the difference in the binding free energy for subsites A, B, E and F and the rate constant of transglycosylation between EGL and HEL.     

细菌几丁质酶基因的表达调控

几丁质酶可以降解几丁质,广泛存在于各类微生物中。几丁质的降解产物几丁寡糖在医药、食品及农业生防领域有很重要的应用价值及广泛的应用前景。细菌在利用几丁质时,需要先分泌几丁质酶,将几丁质降解成几丁寡糖或单体,再通过特异的转运系统送进细胞而被利用。胞内的几丁质降解产物作为特定的信号分子,可以激活或阻遏相应chi基因的转录,从而影响细菌几丁质酶的合成。在各种调节蛋白及应答元件的参与下,细菌几丁质酶的合成受到精密的控制。文章以链霉菌和大肠杆菌为代表综述了细菌在转运系统和基因表达两个层面上控制几丁质酶合成的最新研究进展。     

Expression of a synthetic gene coding for ostrich egg-white lysozyme in Pichia pastoris and its enzymatic activity

To investigate the structure-function relationships of goose-type lysozyme, a gene coding for ostrich egg-white lysozyme (OEL) was designed based on the published amino acid sequence and constructed by assembling 32 chemically synthesized oligonucleotides. To obtain the recombinant OEL (rOEL), the synthetic gene was fused to the alpha-factor signal peptide in the expression vector pPIC9K and expressed in the methylotrophic yeast Pichia pastoris. The secreted protein from the transformed yeast was found to be processed at three different sites, including the correct site. The correctly processed rOEL was purified to homogeneity and shown to be indistinguishable from the authentic form in terms of circular dichroism (CD) spectrum and enzyme activity. Furthermore, the time-course of the reaction catalyzed by OEL was studied using (GlcNAc)(n) (n = 5 and 6) as the substrate and compared to that of goose egg-white lysozyme (GEL) [Honda and Fukamizo (1998) BIOCHIM: Biophys. Acta 1388, 53-65]. OEL hydrolyzed (GlcNAc)(6) in an endo-splitting manner producing mainly (GlcNAc)(2), (GlcNAc)(3), and (GlcNAc)(4), and cleavage to (GlcNAc)(3) + (GlcNAc)(3) predominated over that to (GlcNAc)(2) + (GlcNAc)(4). This indicates that OEL hydrolyzes preferentially the third glycosidic linkage from the nonreducing end of (GlcNAc)(6) as in the case of GEL. The cleavage pattern seen for (GlcNAc)(5) was similar to that seen for (GlcNAc)(6). Theoretical analysis of the reaction time-course for OEL revealed that the binding free energy values for subsites B, E, and G were different between OEL and GEL, although these lysozymes were estimated to have the same type of subsite structure.     

Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue.

The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 A resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is close to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.