Chitoporin from the Marine Bacterium Vibrio harveyi: PROBING THE ESSENTIAL ROLES OF TRP136 AT THE SURFACE OF THE CONSTRICTION ZONE*

VhChiP is a sugar-specific porin present in the outer membrane of the marine bacterium Vibrio harveyi. VhChiP is responsible for the uptake of chitin oligosaccharides, with particular selectivity for chitohexaose. In this study, we employed electrophysiological and biochemical approaches to demonstrate that Trp¹³⁶, located at the mouth of the VhChiP pore, plays an essential role in controlling the channel''s ion conductivity, chitin affinity, and permeability. Kinetic analysis of sugar translocation obtained from single channel recordings indicated that the Trp¹³⁶ mutations W136A, W136D, W136R, and W136F considerably reduce the binding affinity of the protein channel for its best substrate, chitohexaose. Liposome swelling assays confirmed that the Trp¹³⁶ mutations decreased the rate of bulk chitohexaose permeation through the VhChiP channel. Notably, all of the mutants show increases in the off-rate for chitohexaose of up to 20-fold compared with that of the native channel. Furthermore, the cation/anion permeability ratio P_c/P_a is decreased in the W136R mutant and increased in the W136D mutant. This demonstrates that the negatively charged surface at the interior of the protein lumen preferentially attracts cationic species, leading to the cation selectivity of this trimeric channel.     

Molecular Uptake of Chitooligosaccharides through Chitoporin from the Marine Bacterium Vibrio harveyi

Background

Chitin is the most abundant biopolymer in marine ecosystems. However, there is no accumulation of chitin in the ocean-floor sediments, since marine bacteria Vibrios are mainly responsible for a rapid turnover of chitin biomaterials. The catabolic pathway of chitin by Vibrios is a multi-step process that involves chitin attachment and degradation, followed by chitooligosaccharide uptake across the bacterial membranes, and catabolism of the transport products to fructose-6-phosphate, acetate and NH₃.

Principal Findings

This study reports the isolation of the gene corresponding to an outer membrane chitoporin from the genome of Vibrio harveyi. This porin, expressed in E. coli, (so called VhChiP) was found to be a SDS-resistant, heat-sensitive trimer. Immunoblotting using anti-ChiP polyclonal antibody confirmed the expression of the recombinant ChiP, as well as endogenous expression of the native protein in the V. harveyi cells. The specific function of VhChiP was investigated using planar lipid membrane reconstitution technique. VhChiP nicely inserted into artificial membranes and formed stable, trimeric channels with average single conductance of 1.8±0.13 nS. Single channel recordings at microsecond-time resolution resolved translocation of chitooligosaccharides, with the greatest rate being observed for chitohexaose. Liposome swelling assays showed no permeation of other oligosaccharides, including maltose, sucrose, maltopentaose, maltohexaose and raffinose, indicating that VhChiP is a highly-specific channel for chitooligosaccharides.

Conclusion/Significance

We provide the first evidence that chitoporin from V. harveyi is a chitooligosaccharide specific channel. The results obtained from this study help to establish the fundamental role of VhChiP in the chitin catabolic cascade as the molecular gateway that Vibrios employ for chitooligosaccharide uptake for energy production.     

Effects of H-bonds on sugar binding to chitoporin from Vibrio harveyi

BackgroundVhChiP is a sugar-specific-porin present in the outer membrane of the marine bacterium Vibrio harveyi and responsible for chitin uptake, with a high selectivity for chitohexaose.MethodsVhChiP and its mutants were expressed and purified from BL21 (DE3) Omp8 Rosetta strain. After reconstitution into planar lipid bilayers, the ion current fluctuations caused by chitohexaose entering the channel were measured in deuterium oxide and in water.ResultsThe role of hydrogen-bonding in sugar binding was investigated by comparing channel occlusion by chitohexaose in buffers containing H₂O and D₂O. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of VhChiP. Replacing H₂O as solvent by D₂O significantly decreased the on- and off-rates of sugar penetration into the channel. The importance of hydrogen bonding inside the channel was more noticeable when the hydrophobicity of the constriction zone was diminished by replacing Trp¹³⁶ with the charged residues Asp or Arg. The on- and off-rates decreased up to 2.5-fold and 4-fold when Trp¹³⁶ was replaced by Arg, or 5-fold and 3-fold for Trp¹³⁶ replacement by Asp, respectively. Measuring the on-rate at different temperatures and for different channel mutants revealed the activation energy for chitohexaose entrance into VhChiP channel.ConclusionsHydrogen-bonds contribute to sugar permeation.     

Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect β-N-acetyl-D-hexosaminidase

The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp⁴⁹⁰, Glu³²⁸, Val³²⁷ in OfHex1, and Trp³⁵⁸, Tyr¹³¹ and Ile¹⁷⁹ in BGlu1) were identified as being conserved in the +1 sugar binding site. OfHex1 Glu³²⁸ together with Trp⁴⁹⁰ was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in K _m for (GlcNAc)₂ and a 42-fold increment in K _i for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu³⁶⁸ and Asp³⁶⁷) and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu³²⁸ and Val³²⁷ were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes.     

2-Acylamido Analogues of N-Acetylglucosamine Prime Formation of Chitin Oligosaccharides by Yeast Chitin Synthase 2

Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N′-diacetylchitobiose (GlcNAc₂) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc₂ and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc₂ as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.     

Probing the physiological roles of the extracellular loops of chitoporin from Vibrio campbellii

VhChiP, a sugar-specific porin found on the outer membrane of Vibrio campbellii, is responsible for the transport of chitooligosaccharides, allowing the bacterium to thrive in aquatic environments using chitin as a nutrient. We previously showed that VhChiP is composed of three identical subunits, each containing a 16-stranded β-barrel connected by eight extracellular loops and eight short periplasmic turns. This study is focused on the specific roles of three prominent extracellular loops of VhChiP-L2, L3, and L8. The deletion of L2 completely disrupted the L2-L2 interactions, thus destabilizing the protein trimers as well as the integrity of the secondary structure. The deletion of L3 caused a drastic loss in the binding affinity for sugar substrates because of the absence of a cluster of key amino acid residues that form the affinity sites. The removal of L8 induced pronounced gating, which is highly responsive to elevated potentials. Our data provide further information on the important roles of the three prominent loops of VhChiP: loop L2 maintains the trimeric structure and the integrity of secondary structure, loop L3 controls the binding affinity for sugar substrates, and loop L8 retains the stably open state of the channel.     

Site-directed alkylation of LacY: effect of the proton electrochemical gradient

Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H⁺ electrochemical gradient (Δ_μ¯H+, interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Δ_μ¯H+, like sugar, acts to increase the probability of opening on the periplasmic side of LacY.     

Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel,hHV1

Part of the “signature sequence” that defines the voltage-gated proton channel (H_V1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H_V1 genes. Replacing Trp²⁰⁷ in human H_V1 (hH_V1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, E_a, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp²⁰⁷ establishes the major energy barrier between closed and open hH_V1. Cation–π interaction between Trp²⁰⁷ and Arg²¹¹ evidently latches the channel closed. Trp²⁰⁷ mutants lost proton selectivity at pH_o >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H_V1 that is essential to its biological functions, was compromised. In the WT hH_V1, ΔpH-dependent gating is shown to saturate above pH_i or pH_o 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp²⁰⁷ mutants, ΔpH-dependent gating saturated at lower pH_o but not at lower pH_i. That Trp²⁰⁷ mutation selectively alters pH_o sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H_V1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pH_o and pH_i in H_V1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.     

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline,Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate

4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)₄, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with K_i values of 2.01 × 10⁻⁵ and 1.84 × 10⁻⁶ m, respectively. From ITC analysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔH_r°), which is similar to those obtained for 2 and (GlcNAc)₄. However, the entropy loss (−TΔS_r°) for the binding of 3 was smaller than those of 2 and (GlcNAc)₄. Thus the binding of 3 was found to be more favorable than those of the others. Judging from the K_d value of 3 (760 nm), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites −4 to −1 and the moranoline moiety adopts an undistorted ⁴C₁ chair conformation almost overlapping with the −1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835–838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.     

Characterization of DegQVh, a Serine Protease and a Protective Immunogen from a Pathogenic Vibrio harveyi Strain

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ_Vh), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ_Vh was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQ_Vh in T4 was modulated by temperature, possibly through the σ^E-like factor. Enzymatic analyses demonstrated that the recombinant DegQ_Vh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQ_Vh protein were 50°C and pH 8.0. A vaccination study indicated that the purified recombinant DegQ_Vh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQ_Vh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQ_Vh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular β-agarase. The E. coli strain harboring pAQ1 could express and secrete the chimeric DegQ_Vh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ_Vh significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.     

Transfer of Free Polymannose-type Oligosaccharides from the Cytosol to Lysosomes in Cultured Human Hepatocellular Carcinoma HEPG2 Cells

Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H–like enzyme and a mannosidase to yield the linear isomer of Man₅GlcNAc (Man[α1-2]Man[α1-2]Man[α1-3][Man α1-6]Man[β14]GlcNAc). Here we have examined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse–chase incubations with d-[2- ³H]mannose followed by permeabilization of cells with streptolysin O free oligosaccharides were isolated from the resulting cytosolic and membrane-bound compartments. Control pulse–chase experiments revealed that total cellular free oligosaccharides are lost from HepG2 cells with a half-life of 3–4 h. In contrast use of the vacuolar H⁺/ATPase inhibitor, concanamycin A, stabilized total cellular free oligosaccharides and enabled us to demonstrate a translocation of partially trimmed oligosaccharides from the cytosol into a membrane-bound compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 μM swainsonine, which provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels which lead to the accumulation of the linear isomer of Man₅GlcNAc in the cytosol. Subcellular fractionation studies on Percoll density gradients revealed that the cytosol-generated linear isomer of Man₅GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes.     

Functional expression and characterization of a chitinase from the marine archaeon Halobacterium salinarum CECT 395 in Escherichia coli

The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli. The protein called HsChiA1p has a modular structure consisting of a glycosyl hydrolase family 18 catalytic region, as well as a N-terminal family 5 carbohydrate-binding module and a polycystic kidney domain. The purified recombinant chitinase displayed optimum catalytic activity at pH 7.3 and 40 °C and showed high stability over broad pH (6–8.5) and temperature (25–45 °C) ranges. Protein activity was stimulated by the metal ions Mg⁺², K⁺, and Ca⁺² and strongly inhibited by Mn⁺². HsChiA1p is salt-dependent with its highest activity in the presence of 1.5 M of NaCl, but retains 20 % of its activity in the absence of salt. The recombinant enzyme hydrolysed p-NP-(GlcNAc)₃, p-NP-(GlcNAc), crystalline chitin, and colloidal chitin. From its sequence features and biochemical properties, it can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products.     

Cooperative binding ensures the obligatory melibiose/Na+ cotransport in MelB

MelB catalyzes the obligatory cotransport of melibiose with Na⁺, Li⁺, or H⁺. Crystal structure determination of the Salmonella typhimurium MelB (MelB_St) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na⁺ and melibiose has been previously established. To determine why cotranslocation of sugar solute and cation is obligatory, we analyzed each binding in the thermodynamic cycle using three independent methods, including the determination of melting temperature by circular dichroism spectroscopy, heat capacity change (ΔC_p), and regulatory phosphotransferase EIIA^Glc binding with isothermal titration calorimetry (ITC). We found that MelB_St thermostability is increased by either substrate (Na⁺ or melibiose) and observed a cooperative effect of both substrates. ITC measurements showed that either binary formation yields a positive sign in the ΔC_p, suggesting MelB_St hydration and a likely widening of the periplasmic cavity. Conversely, formation of a ternary complex yields negative values in ΔCp, suggesting MelB_St dehydration and cavity closure. Lastly, we observed that EIIA^Glc, which has been suggested to trap MelB_St at an outward-open state, readily binds to the MelB_St apo state at an affinity similar to MelB_St/Na⁺. However, it has a suboptimal binding to the ternary state, implying that MelB_St in the ternary complex may be conformationally distant from the EIIA^Glc-preferred outward-facing conformation. Our results consistently support the notion that binding of one substrate (Na⁺ or melibiose) favors MelB_St at open states, whereas the cooperative binding of both substrates triggers the alternating-access process, thus suggesting this conformational regulation could ensure the obligatory cotransport.     

Purification and Characterization of Three Thermostable Endochitinases of a Noble Bacillus Strain, MH-1, Isolated from Chitin-Containing Compost

A thermophilic and actinic bacterium strain, MH-1, which produced three different endochitinases in its culture fluid was isolated from chitin-containing compost. The microorganism did not grow in any of the usual media for actinomyces but only in colloidal chitin supplemented with yeast extract and (2,6-O-dimethyl)-β-cyclodextrin. Compost extract enhanced its growth. In spite of the formation of branched mycelia, other properties of the strain, such as the formation of endospores, the presence of meso-diaminopimelic acid in the cell wall, the percent G+C of DNA (55%), and the partial 16S ribosomal DNA sequence, indicated that strain MH-1 should belong to the genus Bacillus. Three isoforms of endochitinase (L, M, and S) were purified to homogeneity and characterized from Bacillus sp. strain MH-1. They had different molecular masses (71, 62, and 53 kDa), pIs (5.3, 4.8, and 4.7), and N-terminal amino acid sequences. Chitinases L, M, and S showed relatively high temperature optima (75, 65, and 75°C) and stabilities and showed pH optima in an acidic range (pH 6.5, 5.5, and 5.5, respectively). When reacted with acetylchitohexaose [(GlcNAc)₆], chitinases L and S produced (GlcNAc)₂ at the highest rate while chitinase M produced (GlcNAc)₃ at the highest rate. None of the three chitinases hydrolyzed (GlcNAc)₂. Chitinase L produced (GlcNAc)₂ and (GlcNAc)₃ in most abundance from 66 and 11% partially acetylated chitosan. The p-nitrophenol (pNP)-releasing activity of chitinase L was highest toward pNP-(GlcNAc)₂, and those of chitinases M and S were highest toward pNP-(GlcNAc)₃. All three enzymes were inert to pNP-GlcNAc. AgCl, HgCl₂, and (GlcNAc)₂ inhibited the activities of all three enzymes, while MnCl₂ and CaCl₂ slightly activated all of the enzymes.     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

Characteristics of cold-adaptive endochitinase from Antarctic bacterium Sanguibacter antarcticus KOPRI 21702

The psychrotrophic Sanguibacter antarcticus KOPRI 21702^T, isolated from Antarctic seawater, produced a cold-adapted chitinolytic enzyme that is a new 55 kDa family 18 chitinase (Chi21702). Chi21702 exhibited high activities toward pNP-(GlcNAc)₂ and pNP-(GlcNAc)₃ with no activity for pNP-GlcNAc, indicating that it prefers chitin chains longer than dimers, just as endochitinases do. A mixture of GlcNAc and GlcNAc₂ was produced as a main product by Chi21702 activity from chitin oligosaccharides and swollen chitin, while less GlcNAc₃ was produced. These results show that Chi21702 has an endochitinase activity, randomly hydrolyzing chitin at internal sites. Chi21702 displayed chitinase activity at 0–40 °C (optimal temperature of 37 °C), maintained its activity at pH 4–11 (optimal pH of 7.6). Interestingly, Chi21702 exhibited relative activities of 40% and 60% at 0 and 10 °C, respectively, in comparison to 100% at 37 °C, which is higher than those of the previously characterized, cold-adapted, chitinases from bacterial strains.     

Enhanced Amino Acid Selection in Fully Evolved Tryptophanyl-tRNA Synthetase,Relative to Its Urzyme,Requires Domain Motion Sensed by the D1 Switch,a Remote Dynamic Packing Motif

We previously showed (Li, L., and Carter, C. W., Jr. (2013) J. Biol. Chem. 288, 34736–34745) that increased specificity for tryptophan versus tyrosine by contemporary Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) over that of TrpRS Urzyme results entirely from coupling between the anticodon-binding domain and an insertion into the Rossmann-fold known as Connecting Peptide 1. We show that this effect is closely related to a long range catalytic effect, in which side chain repacking in a region called the D1 Switch, accounts fully for the entire catalytic contribution of the catalytic Mg²⁺ ion. We report intrinsic and higher order interaction effects on the specificity ratio, (k_cat/K_m)_Trp/(k_cat/K_m)_Tyr, of 15 combinatorial mutants from a previous study (Weinreb, V., Li, L., and Carter, C. W., Jr. (2012) Structure 20, 128–138) of the catalytic role of the D1 Switch. Unexpectedly, the same four-way interaction both activates catalytic assist by Mg²⁺ ion and contributes −4.4 kcal/mol to the free energy of the specificity ratio. A minimum action path computed for the induced-fit and catalytic conformation changes shows that repacking of the four residues precedes a decrease in the volume of the tryptophan-binding pocket. We suggest that previous efforts to alter amino acid specificities of TrpRS and glutaminyl-tRNA synthetase (GlnRS) by mutagenesis without extensive, modular substitution failed because mutations were incompatible with interdomain motions required for catalysis.     

Histidine 352 (His352) and Tryptophan 355 (Trp355) Are Essential for Flax UGT74S1 Glucosylation Activity toward Secoisolariciresinol

Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the Arabidopsis thaliana UGT72B1 model. The ligand docking predicted Ser³⁵⁷ and Trp³⁵⁵ as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys³³⁵, Gln³³⁷ and Trp³⁵⁵ were predicted to bind the 7-OH, 2-OCH₃ and 17-OCH₃ of SECO. Site-directed mutagenesis of Cys³³⁵, Gln³³⁷, His³⁵², Trp³⁵⁵ and Ser³⁵⁷_, and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp³⁵⁵ was substituted to Ala³⁵⁵ and Gly³⁵⁵ or when changing His³⁵² to Asp³⁵²_, and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp³⁵⁵ and His³⁵² are critical for UGT74S1’s glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.     

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2_hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2_hPg. However, the binding strength of VEK30 (K_D = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (K_D = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH₂ and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2_hPg with nearly the same affinity as PAM (K_D = 1–2 nm). However, addition of two PAM residues (Arg¹²⁶-His¹²⁷) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2_hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg¹¹³, His¹¹⁴, Glu¹¹⁶, Arg¹²⁶, His¹²⁷), mutation of which reduced PAM binding affinity for K2_hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.