Báo cáo khoa học: Crystal structure of a cold-adapted class C b-lactamase

Crystal structure of a cold-adapted class C β-lactamase

Tác giả: Catherine Michaux, Jan Massant, Frédéric Kerff, Jean-Marie Frère, Jean-Denis Docquier, Isabel Vandenberghe, Bart Samyn, Annick Pierrard, Georges Feller, Paulette Charlier, Jozef Van Beeumen, Johan Wouters

Lĩnh vực: Sinh học cấu trúc phân tử, Hóa sinh, Kỹ thuật protein

Nội dung tài liệu: Nghiên cứu này trình bày cấu trúc tinh thể của enzyme β-lactamase nhóm C thích nghi với nhiệt độ thấp từ vi khuẩn ưa lạnh *Pseudomonas fluorescens*. Với độ phân giải 2.2 Å, đây là một trong số ít các cấu trúc tinh thể của protein ưa lạnh được xác định. Cấu trúc này được so sánh với các enzyme tương đồng thích nghi với nhiệt độ trung bình và một protein ưa lạnh khác được mô hình hóa. Việc làm sáng tỏ cấu trúc 3D của enzyme này cung cấp những hiểu biết sâu sắc hơn về các đặc điểm liên quan đến sự thích nghi với nhiệt độ thấp. So sánh cấu trúc giữa β-lactamase ưa lạnh và ưa nhiệt cho thấy rằng lực tĩnh điện đóng vai trò quan trọng trong sự thích nghi ở nhiệt độ thấp, với số lượng tương tác ion thấp hơn ở các enzyme ưa lạnh. Các enzyme ưa lạnh còn đặc trưng bởi số lượng liên kết hydro giảm, hàm lượng proline thấp hơn và tỷ lệ arginine so với lysine thấp hơn. Tất cả những đặc điểm này làm cho cấu trúc trở nên linh hoạt hơn, cho phép enzyme hoạt động hiệu quả ở nhiệt độ thấp.

Mục lục chi tiết:

• Keywords
• Correspondence
• Database
• In this study, the crystal structure of a class C β-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 Å resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic β-lactamases shows that electrostatics seems to play a major role in low-temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures.
• β-Lactamases are the major causes of bacterial resistance to the β-lactam family of antibiotics, such as penicillins and cephalosporins. These enzymes catalyze the hydrolysis of the critical β-lactam ring and render the antibiotic inactive against its original cellular target, the cell wall transpeptidase. β-Lactamases of
• Abbreviations
• Psychrophilic class C β-lactamase
• Classes A, C and D are active site serine enzymes, whereas class B β-lactamases require one or two zinc ions for their activity [1]. Only class C β-lactamases were found to be synthesized by ampicillin-resistant psychrophilic bacteria collected in the Antarctic [2]. Psychrophilic strains, and particularly their enzymes, have generated considerable interest and have been proposed for a number of applications in fundamental research [3,4], in biotechnology to improve the efficiency of industrial processes, and for environmental applications [5–7]. ‘Cold enzymes’ from psychrophilic microorganisms are generally characterized by a higher catalytic activity and efficiency (kcat/Km) at low temperatures than their mesophilic counterparts [8]. The ability of psychrophilic microorganisms to survive and proliferate at low temperatures implies that they have overcome key barriers inherent to permanently cold environments, such as protein cold-denaturation, inappropriate protein folding, and reduced enzyme activity, to name a few [9]. The commonly accepted hypothesis for this cold adaptation is the activity-stability-flexibility relationship, which suggests that psychrophilic enzymes increase the flexibility of their structures to compensate for the ‘freezing effect’ of cold habitats [8,10–14]. Increased intramolecular flexibility is achieved through weakening of interactions that stabilize the native protein molecules, especially those involved in catalysis, with a concomitant reduction in stability of cold-adapted enzymes [15,16]. A general theory for cold adaptation has not been formulated yet, as different enzymatic families can follow different evolutionary strategies. Therefore, recently, the research community has focused on comparative structural investigations of homologous proteins adapted to different temperature conditions [17–25]. In contrast to thermophilic proteins, few crystal structures have been solved for psychrophilic proteins, probably because their thermolability and flexibility result in handling and crystallization difficulties [26]. Analysis of the available 3D structures and site-directed mutagenesis experiments has shown that the low stability of cold-adapted enzymes has been achieved through: a reduction of the number and/or strength of weak interactions; increased interactions with the solvent; a decrease in the number and/or strength of hydrophobic internal clusters; and entropic effects tending to increase the entropy of the unfolded form and to lower its free energy. Each cold-adapted enzyme is modulated using a specific strategy, probably as a function of structural requirements, and makes a selection among the above-mentioned factors to improve the flexibility at the level of the catalytic site [27]. In this work, we describe the crystal structure of a psychrophilic class C β-lactamase from Pseudomonas fluorescens TAE4 [2] and compare its structure to those of three homologs produced by the psychrophile Psychrobacter immobilis [28] and the two mesophiles Enterobacter cloacae 908R and Serratia marcescens [29]. These enzymes were selected because of their availability for experimental assays. The 3D structure of the homologs was modeled, as no structure was available in the Protein Data Bank, except for the mesophile 908R (Protein Data Bank entry 1Y54). The comparison of these structures of psychrophilic enzymes with those of mesophilic counterparts with high sequence identity provides further insights into the understanding of cold adaptation.
• Results
• Kinetic characterization of the cold enzyme from Pse. fluorescens TAE4
• Kinetic parameters for the hydrolysis of three cephalosporins (nitrocefin, cephalexin, and cefazolin) and five penicillins (benzylpenicillin, ampicillin, carbenicillin, oxacillin, and cloxacillin) were determined for the TAE4 β-lactamase and compared with those of the enzymes from Psy. immobilis, E. cloacae 908R and S. marcescens (Table 1). The substrate profile of the TAE4 β-lactamase is globally similar to that of its psychrophilic and mesophilic homologs, except for penicillins with larger side chains (oxacillin and cloxacillin) and carbenicillin. The latter are very poor substrates of mesophilic class C β-lactamases. The kcat values measured for Pse. fluorescens are 26–130 times higher than those of E. cloacae 908R. As the Km values are also higher (lower apparent affinity), the kcat/Km ratios are similar for both enzymes. These data probably result from a difference in the deacylation rates between the enzymes.
• Stability and thermal and urea denaturation of the cold enzyme from Pse. fluorescens TAE4
• Thermal inactivation of β-lactamases from Pse. fluorescens, Psy. immobilis A5, S. marcescens and E. cloacae was studied at one or different temperatures following fluorescence quenching (Table 2). The thermal denaturation is irreversible for the four proteins, and therefore only kinetic parameters can be deduced. Both cold enzymes (Pse. fluorescens, Psy. immobilis) are more sensitive to thermal denaturation than their mesophilic homologs. At 50 °C, the measured ka values for both Antarctic enzymes are 22-60 times larger than that of S. marcescens. Intrinsic fluorescence of the psychrophile TAE4 was also measured as a function of the urea concentration at 30 °C (Fig. 1). As denaturation of Pse. fluorescens TAE4 by urea is nearly fully reversible (more than 95%), thermodynamic parameters can be deduced. The Cm, the slope of the line relating the free energy difference between the native (N) and denatured (D) form at a given urea concentration to the urea concentration (MD_N) and the free energy difference between the native (N) and the denatured (D) form without denaturing agent (AG°D-N) were 2.4 M urea, 3.7 kcal·mol⁻¹·M⁻¹ and 8.7 kcal·mol⁻¹, respectively. The thermodynamic stability of TAE4 is lower than that of the mesophilic enzyme, AmpC, as the AG°D-N value is 5.3 times smaller for the cold enzyme [30].
• Sequence comparison
• The complete amino acid sequence of the psychrophile TAE4 was determined using analyses carried out on the protein itself. With exception of the dipeptide Leu83-Lys84, which was lost during purification of the
• Lys-C protease-generated peptides, all other amino acids could be identified in at least one of the peptides generated by the three proteases used. The summed molecular masses of the subsequent Lys-C peptides was 38 720.2 Da, which agrees with the experimentally determined mass of the protein of 38 723.1 Da (± 4.9 Da). The converted spectrum reveals a shoulder at a mass of around 38 700 Da, which reflects a one-residue heterogeneity detected by chemical C-terminal sequence analysis (-SAMDQ and -SAMD).
• The four studied β-lactamases, aligned using CLUSTALW, share an amino acid sequence identity of about 40-50% (Fig. 2 and Table 3). The C-terminal region is relatively conserved, whereas the N-terminal region is the most variable (data not shown). They share the three characteristic motifs of serine-reactive β-lactamases [31,32]: S-X-X-K, with Ser64 and Lys67 forming hydrogen bonds in the active site, Y-X-N, with Tyr150 and Asn152 pointing into the active site, and KTG (Lys315), forming the opposite wall of the active site
• Fig. 2. Sequence alignment of class C β-lactamases from Pse. fluorescens (PSEFL), Psy. immobilis (PSYIM), E. cloacae (ENTCL) and S. marcescens (SERMA). The three motifs characteristic of active site serine β-lactamases are in red. The disordered sequences of PSEFL are in green.
• Table 3. Percentage identity between the four β-lactamases and rmsd values (Å) for Cα atoms among the four studied enzymes. Parameters potentially involved in thermal adaptation. ASA, accessible surface area.
• Fig. 3. Crystal structure of the class C β-lactamase TAE4 from Pse. fluorescens. The important residues of the active site are labeled. The unobserved loop is indicated by the black box.
• (Protein Data Bank code 1FR1, 2.0 Å) or E. cloacae P99 (Protein Data Bank code 1XX2, 1.88 Å). They share 35.6% and 43.1% sequence identities, respectively, with their templates, and the obtained models are reliable as indicated by the Ramachandran plot (data not shown). These different model structures and the crystal structures of the psychrophilic enzyme TAE4 from Pse. fluorescens (Protein Data Bank code 2QZ6, 2.2 Å) and the mesophilic homolog from E. cloacae 908R (Protein Data Bank code 1Y54, 2.1 Å) were compared in order to identify the interactions and the structural features potentially involved in the low stability and structural flexibility of the psychrophilic enzymes. The overall folding is identical for all the enzymes. Superimposition of the four proteins shows quite low rmsd values (Table 3). Moreover, the conformations of the catalytic triad and the specificity pocket are very similar. Only subtle modifications of the enzyme conformation therefore account for the low stability of cold enzymes. In this context, the disordered region, not observed in the crystal structure of the 908R enzyme and close to the catalytic pocket, is assumed to
• Table 4. Data collection and refinement statistics for P. fluorescens β-lactamase TAE4. Values listed in parentheses are for the highest resolution (2.3–2.2 Å). Rfactor = 2|| Fo| – |FC||/Σ|Fo Rfree was calculated with 5% of the reflections set aside randomly throughout the refinement.
• be partly responsible for the larger flexibility of the cold β-lactamase and for its ability to hydrolyze large substrates. Both psychrophilic β-lactamases have fewer ion pairs (3) than the mesophilic ones (5 and 11), showing that electrostatic effects may play a role in the stability of the latter proteins (Table 3). One salt bridge (Glu272-Arg148) involving residues close to the active site is conserved among the four enzymes and probably contributes to the activity. The ion pairs present in the mesophilic but absent in the psychrophilic enzymes are distributed throughout the whole structure. In addition, the number of hydrogen bonds between side chains is also slightly smaller in the case of cold β-lactamases. Even though the mesophilic enzymes have more hydrophobic residues, the hydrophobic contacts and aromatic interactions are similar in all enzymes. Frequently, alterations of the accessible surface of nonpolar side chains and of the accessible charged surfaces are observed in cold-adapted enzymes. Polar and apolar accessible surface areas were therefore also calculated, but they seem to be not correlated with thermal stability in the present cases.
• Discussion The determination of the crystal structure of a psychrophilic class C β-lactamase, from Pse. fluorescens TAE4, and its comparison with one psychrophilic and two mesophilic homologs, allowed a detailed structural analysis to obtain insights into features involved in cold adaptation. Although the four proteins have a very similar fold that is characteristic of class C β-lactamases, subtle sequence and structure differences could be seen. No significant differences in the number and nature of residues were observed around the active site (within 12 Å from the catalytic serine) of the four proteins. However, one loop at one edge of the active site (Glu123-Asn127) of the Pse. fluorescens β-lactamase was undetectable in the electron density map and was therefore assumed to be disordered, which is not the case for the mesophilic homolog 908R. This flexibility, in spite of the steric hindrance of the substrate, is thought to be partially responsible for its unexpected activity on large penicillin substrates. The kinetic parameters of the Pse. fluorescens β-lactamase unambiguously show that the psychrophilic enzyme is more active on large substrates (26-130 times), although the active site structure and composition are identical to those of mesophilic β-lactamases. This indicates that the active site of the Pse. fluorescens β-lactamase is more easily accessible to large substrates and should
• be more dynamic in solution, i.e. flexible in a broad sense. Furthermore, the higher Km (lower apparent affinity) also suggests a more mobile active site that binds the substrates weakly. In addition, the more flexible conformation of Pse. fluorescens β-lactamase would allow easier access of the water molecule in the active site of the enzyme, accelerating the deacylation. These assumptions would also explain the low thermal and chemical stability of the enzyme. It should be noted that these results parallel those obtained for a psychrophilic α-amylase, the latter showing higher activity on large and branched polysaccharides, with, however, a higher Km, when compared with a mesophilic homolog [33]. Moreover, electrostatics seems to play a major role in the cold adaptation of the present β-lactamases. Indeed, the cold enzymes have a lower total number of ionic interactions than the mesophilic ones. Even though the differences may not appear to be dramatic (only two between the S. marcescens enzyme and the cold enzymes), it has already been shown that a single ion pair difference can reflect adaptation to low or high temperatures [23,34]. A strong correlation was also found between thermal stability and the content of basic residues. The psychrophilic enzymes have a slightly lower arginine content and a higher lysine con- tent than their mesophilic homologs, a characteristic of several cold-adapted enzymes [35]. Arginine is a stabilizing residue [36] because of the ability of its guanidinium group to form five hydrogen bonds with surrounding residues, as well as two salt bridges with acidic groups. In addition, lysine residues are more flexible than arginine. Finally, it was also observed that both psychrophilic β-lactamases are overall negatively charged, in contrast to their mesophilic homo- logs, which supports the conclusion that charges and electrostatics are probably involved in the temperature adaptation. Other differences were also observed. Given the mean errors of 0.15 Å on coordinates (Luzzati plot), the psy- chrophilic β-lactamases are characterized by a decreased number of hydrogen bonds, possibly rendering the structure more flexible. To confirm this tendency, higher-resolution structures would be necessary to improve the accuracy of those geometries. In addition, even though the number of hydrophobic contacts is not correlated with the thermostability, the number of hydrophobic aliphatic residues, such as alanine, valine, leucine, and isoleucine, is smaller for the cold β-lacta- mases. Several examples show that hydrophobicity is positively correlated with the thermostability [37–39]. The number of prolines is also slightly lower for both psychrophilic β-lactamases. Prolyl residues can
• adopt only a few conformations and restrict the available dihedral angles of the preceding residue; thus, proline has the lowest conformational entropy and contributes to the local rigidity of the peptidic backbone. Previous crystallographic studies have indicated that, in addition to the features already mentioned, some cold-adapted enzymes may be characterized by a decreased number of disulfide bonds, an increased number of glycine residues, a reduced apolar fraction in the core, higher accessibility of