A serine protease triad forms the catalytic centre of a triacylglycerol lipase

True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.     

Structure of the pancreatic lipase-procolipase complex.

Interfacial adsorption of pancreatic lipase is strongly dependent on the physical chemical properties of the lipid surface. These properties are affected by amphiphiles such as phospholipids and bile salts. In the presence of such amphiphiles, lipase binding to the interface requires a protein cofactor, colipase. We obtained crystals of the pancreatic lipase-procolipase complex and solved the structure at 3.04 A resolution. Here we describe the structure of procolipase, which essentially consists of three 'fingers' and is topologically comparable to snake toxins. The tips of the fingers contain most of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs at the opposite side to this site and involves polar interactions. Determination of the three-dimensional structure of pancreatic lipase has revealed the presence of two domains: an amino-terminal domain, at residues 1-336 containing the active site and a carboxy-terminal domain at residues 337-449 (ref. 6). Procolipase binds exclusively to the C-terminal domain of lipase. No conformational change in the lipase molecule is induced by the binding of procolipase.     

Convergent evolution of similar function in two structurally divergent enzymes

An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase. Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin. Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site. Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other. We have determined the crystal structure of E. coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure. The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system. This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently.     

The molecular evolution of genes and proteins: a tale of two serines

The advent of techniques for cloning and rapidly sequencing DNA has produced an explosive increase of sequence information for nucleic acids and their inferred proteins. Careful study of this large store of data might give us new insights into the relations between the linear sequences of genes and their functions embodied in the three-dimensional structure of proteins, and also illuminate the origin and evolution of the structural complexity of present-day proteins. Here I argue from such a study that the active site sequences of enzymes that have analogous essential serine residues lie in fact on two lines of descent from an ancient ancestral enzyme which had a cysteine instead of serine in its active site. This is based on the assumption that the two codon types which define the separate lines of descent and which have different bases in two positions could not interconvert by single mutations.     

根癌农杆菌介导脂肪酶在无孢黑曲霉中的高效表达

黑曲霉(Aspergillus niger)具有高效的蛋白表达和分泌能力.为实现异源蛋白在无孢黑曲霉中的高效重组表达,在优化遗传筛选标记的基础上,建立了根癌农杆菌介导的、以潮霉素抗性基因为筛选标记的黑曲霉转化体系.利用该体系介导米黑根毛霉脂肪酶(RML)转化黑曲霉SH-1,通过PCR鉴定、酶活检测、镍柱亲和层析及SDS-PAGE电泳检测等确定成功获得了RML的黑曲霉转化子,并通过发酵条件优化实现了RML的高效表达,对硝基苯酚比色法测得转化子酶活最高可达15 U/mL,是其在酵母中表达水平的10倍以上.     

蛋白酶对脂肪酶活性影响的研究

为了弄清洗涤剂中碱性蛋白酶及金属离子对碱性脂肪酶稳定性的问题，对加酶洗涤剂中的碱性蛋白酶及金属离子对圆弧青霉ＰＧ３７所产碱性脂肪酶的活力影响进行了研究。在碱性脂肪酶溶液中分别添加不同量的碱性蛋白酶和金属离子，并在２５℃保温３０ｍｉｎ，分别测定保温前后的脂肪酶活力。结果表明：在一定浓度范围内的蛋白酶对ＰＧ３７碱性脂肪酶活力的影响较小，可同时应用于洗涤剂中；金属离子对脂肪酶活力无明显影响，甚至在一定浓度范围内对酶活有激活作用。总之，ＰＧ３７菌株所产碱性脂肪酶具有良好的酶学特性，非常适合于洗涤剂用酶。     

Structure of human pancreatic lipase

Pancreatic lipase (triacylglycerol acyl hydrolase) fulfills a key function in dietary fat absorption by hydrolysing triglycerides into diglycerides and subsequently into monoglycerides and free fatty acids. We have determined the three-dimensional structure of the human enzyme, a single-chain glycoprotein of 449 amino acids, by X-ray crystallography and established its primary structure by sequencing complementary DNA clones. Enzymatic activity is lost after chemical modification of Ser 152 in the porcine enzyme, indicating that this residue is essential in catalysis, but other data are more consistent with a function in interfacial recognition. Our structural results are evidence that Ser 152 is the nucleophilic residue essential for catalysis. It is located in the larger N-terminal domain at the C-terminal edge of a doubly wound parallel beta-sheet and is part of an Asp-His-Ser triad, which is chemically analogous to, but structurally different from, that in the serine proteases. This putative hydrolytic site is covered by a surface loop and is therefore inaccessible to solvent. Interfacial activation, a characteristic property of lipolytic enzymes acting on water-insoluble substrates at water-lipid interfaces, probably involves a reorientation of this flap, not only in pancreatic lipases but also in the homologous hepatic and lipoprotein lipases.     

脂肪酶作用下鱼油和卵磷脂的酯交换反应

在正己烷有机溶剂中脂肪酶能有效地催化鱼油和卵磷脂的酯交换反应,本研究探讨了反应温度,酶活力和底物不同比等条件下对卵磷脂和鱼油在的ω－3脂肪酸结合率的影响,结果表明,当在25mL的正己烷中,反应温度为45度,酶活力为0．8u/mL,r(卵磷脂）：r( 鱼油）＝1：4时,反应24h后卵磷脂中DHA／EPA的结合率为16．3％。     

Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis

Beta-Lactamases (EC 3.5.2.6, 'penicillinases') are a family of enzymes that protect bacteria against the lethal effects of cell-wall synthesis of penicillins, cephalosporins and related antibiotic agents, by hydrolysing the beta-lactam antibiotics to biologically inactive compounds. Their production can, therefore, greatly contribute to the clinical problem of antibiotic resistance. Three classes of beta-lactamases--A, B and C--have been identified on the basis of their amino-acid sequence; class B beta-lactamases are metalloenzymes, and are clearly distinct from members of class A and C beta-lactamases, which both contain an active-site serine residue involved in the formation of an acyl enzyme with beta-lactam substrates during catalysis. It has been predicted that class C beta-lactamases share common structural features with D,D-carboxypeptidases and class A beta-lactamases, and further, suggested that class A and class C beta-lactamases have the same evolutionary origin as other beta-lactam target enzymes. We report here the refined three-dimensional structure of the class C beta-lactamase from Citrobacter freundii at 2.0-A resolution and confirm the predicted structural similarity. The refined structure of the acyl-enzyme formed with the monobactam inhibitor aztreonam at 2.5-A resolution defines the enzyme's active site and, along with molecular modelling, indicates a mechanism for beta-lactam hydrolysis. This leads to the hypothesis that Tyr 150 functions as a general base during catalysis.     

Ser-His-Glu triad forms the catalytic site of the lipase from Geotrichum candidum

The Ser-His-Asp triad is a well known structural feature of the serine proteases. It has also been directly observed in the catalytic sites of two lipases, whose high-resolution three-dimensional structures have been determined 1,2. Lipases show a wide variety of sizes, substrate and positional specificities, and catalytic rates 3. They achieve maximal catalytic rates at oil-water interfaces. The fungus Geotrichum candidum produces several different forms of lipases, two of which have been purified to homogeneity 4,5. Two lipase genes have been identified, cloned and sequenced 6,7. Both code for proteins of 544 amino acids with a total relative molecular mass of about 60,000 (Mr 60K). The two forms are 86% identical. Their isoelectric points differ slightly, being between 4.3 and 4.6. About 7% of the total Mr is carbohydrate. Until now, only a low resolution structure of GCL has been reported 8, but no high resolution structure has followed. We now report the three-dimensional structure of a lipase from G. candidum (GCL) at 2.2 A resolution. Unlike the other lipases and serine proteases, the catalytic triad of GCL is Ser-His-Glu, with glutamic acid replacing the usual aspartate. Although the sequence similarity with the other two lipases is limited to the region near the active-site serine, there is some similarity in their three-dimensional structures. The GCL is also an alpha/beta protein with a central mixed beta sheet whose topology is similar to that of the N-terminal domain of human pancreatic lipase. As in the other lipases 1,2, the catalytic site is buried under surface loops. Sequence comparisons with proteins from the cholinesterase family suggest that they also contain the Ser-His-Glu triad.     

Structure and catalytic mechanism of the human histone methyltransferase SET7/9

Acetylation, phosphorylation and methylation of the amino-terminal tails of histones are thought to be involved in the regulation of chromatin structure and function. With just one exception, the enzymes identified in the methylation of specific lysine residues on histones (histone methyltransferases) belong to the SET family. The high-resolution crystal structure of a ternary complex of human SET7/9 with a histone peptide and cofactor reveals that the peptide substrate and cofactor bind on opposite surfaces of the enzyme. The target lysine accesses the active site of the enzyme and the S-adenosyl-l-methionine (AdoMet) cofactor by inserting its side chain into a narrow channel that runs through the enzyme, connecting the two surfaces. Here we show from the structure and from solution studies that SET7/9, unlike most other SET proteins, is exclusively a mono-methylase. The structure indicates the molecular basis of the specificity of the enzyme for the histone target, and allows us to propose a model for the methylation reaction that accounts for the role of many of the residues that are invariant across the SET family.     

Structural basis for gate-DNA recognition and bending by type IIA topoisomerases

Type II topoisomerases disentangle DNA to facilitate chromosome segregation, and represent a major class of therapeutic targets. Although these enzymes have been studied extensively, a molecular understanding of DNA binding has been lacking. Here we present the structure of a complex between the DNA-binding and cleavage core of Saccharomyces cerevisiae Topo II (also known as Top2) and a gate-DNA segment. The structure reveals that the enzyme enforces a 150 degrees DNA bend through a mechanism similar to that of remodelling proteins such as integration host factor. Large protein conformational changes accompany DNA deformation, creating a bipartite catalytic site that positions the DNA backbone near a reactive tyrosine and a coordinated magnesium ion. This configuration closely resembles the catalytic site of type IA topoisomerases, reinforcing an evolutionary link between these structurally and functionally distinct enzymes. Binding of DNA facilitates opening of an enzyme dimerization interface, providing visual evidence for a key step in DNA transport.     

The structural basis of Holliday junction resolution by T7 endonuclease I

The four-way (Holliday) DNA junction is the central intermediate in homologous recombination, a ubiquitous process that is important in DNA repair and generation of genetic diversity. The penultimate stage of recombination requires resolution of the DNA junction into nicked-duplex species by the action of a junction-resolving enzyme, examples of which have been identified in a wide variety of organisms. These enzymes are nucleases that are highly selective for the structure of branched DNA. The mechanism of this selectivity has, however, been unclear in the absence of structural data. Here we present the crystal structure of the junction-resolving enzyme phage T7 endonuclease I in complex with a synthetic four-way DNA junction. Although the enzyme is structure-selective, significant induced fit occurs in the interaction, with changes in the structure of both the protein and the junction. The dimeric enzyme presents two binding channels that contact the backbones of the junction's helical arms over seven nucleotides. These interactions effectively measure the relative orientations and positions of the arms of the junction, thereby ensuring that binding is selective for branched DNA that can achieve this geometry.     

Molecular structure of an aspartic proteinase zymogen, porcine pepsinogen, at 1.8 A resolution

The only well-understood mechanism of zymogen activation is that of the serine proteinases, in which proteolytic cleavage leads to conformational changes resulting in a functional active site. A different mechanism is now unveiled by the crystal structure of pepsinogen. Salt bridges that stabilize the positioning of the N-terminal proenzyme segment across the active site of pepsin are disrupted at low pH, releasing the amino-terminal segment and thereby exposing the catalytic apparatus and the substrate-binding sites.     

Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent.

Lipases belong to a class of esterases whose activity on triglycerides is greatly enhanced at lipid-water interfaces. This phenomenon, called interfacial activation, has a structural explanation: a hydrophobic lid, which at rest covers the catalytic site, is displaced on substrate or inhibitor binding and probably interacts with the lipid matrix. Fusarium solani pisi cutinase belongs to a group of homologous enzymes of relative molecular mass 22-25K (ref. 7) capable of degrading cutin, the insoluble lipid-polyester matrix covering the surface of plants, and hydrolysing triglycerides. Cutinases differ from classical lipases in that they do not exhibit interfacial activation; they are active on soluble as well as on emulsified triglycerides. Cutinases therefore establish a bridge between esterases and lipases. We report here the three-dimensional structure of a recombinant cutinase from F. solani pisi, expressed in Escherichia coli. Cutinase is an alpha-beta protein; the active site is composed of the triad Ser 120, His 188 and Asp 175. Unlike other lipases, the catalytic serine is not buried under surface loops, but is accessible to solvent. This could explain why cutinase does not display interfacial activation.     

Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor.

Nerve growth factor (NGF) is involved in a variety of processes involving signalling, such as cell differentiation and survival, growth cessation and apoptosis of neurons. These events are mediated by NGF as a result of binding to its two cell-surface receptors, TrkA and p75. TrkA is a receptor with tyrosine kinase activity that forms a high-affinity binding site for NGF. Of the five domains comprising its extracellular portion, the immunoglobulin-like domain proximal to the membrane (TrkA-d5 domain) is necessary and sufficient for NGF binding. Here we present the crystal structure of human NGF in complex with human TrkA-d5 at 2.2 A resolution. The ligand-receptor interface consists of two patches of similar size. One patch involves the central beta-sheet that forms the core of the homodimeric NGF molecule and the loops at the carboxy-terminal pole of TrkA-d5. The second patch comprises the amino-terminal residues of NGF, which adopt a helical conformation upon complex formation, packing against the 'ABED' sheet of TrkA-d5. The structure is consistent with results from mutagenesis experiments for all neurotrophins, and indicates that the first patch may constitute a conserved binding motif for all family members, whereas the second patch is specific for the interaction between NGF and TrkA.     

Structure of the sulphiredoxin-peroxiredoxin complex reveals an essential repair embrace

Typical 2-Cys peroxiredoxins (Prxs) have an important role in regulating hydrogen peroxide-mediated cell signalling. In this process, Prxs can become inactivated through the hyperoxidation of an active site Cys residue to Cys sulphinic acid. The unique repair of this moiety by sulphiredoxin (Srx) restores peroxidase activity and terminates the signal. The hyperoxidized form of Prx exists as a stable decameric structure with each active site buried. Therefore, it is unclear how Srx can access the sulphinic acid moiety. Here we present the 2.6 A crystal structure of the human Srx-PrxI complex. This complex reveals the complete unfolding of the carboxy terminus of Prx, and its unexpected packing onto the backside of Srx away from the Srx active site. Binding studies and activity analyses of site-directed mutants at this interface show that the interaction is required for repair to occur. Moreover, rearrangements in the Prx active site lead to a juxtaposition of the Prx Gly-Gly-Leu-Gly and Srx ATP-binding motifs, providing a structural basis for the first step of the catalytic mechanism. The results also suggest that the observed interactions may represent a common mode for other proteins to bind to Prxs.