Structural basis for substrate delivery by acyl carrier protein in the yeast fatty acid synthase

In the multifunctional fungal fatty acid synthase (FAS), the acyl carrier protein (ACP) domain shuttles reaction intermediates covalently attached to its prosthetic phosphopantetheine group between the different enzymatic centers of the reaction cycle. Here, we report the structure of the Saccharomyces cerevisiae FAS determined at 3.1 angstrom resolution with its ACP stalled at the active site of ketoacyl synthase. The ACP contacts the base of the reaction chamber through conserved, charge-complementary surfaces, which optimally position the ACP toward the catalytic cleft of ketoacyl synthase. The conformation of the prosthetic group suggests a switchblade mechanism for acyl chain delivery to the active site of the enzyme.     

Crystal structure of the termination module of a nonribosomal peptide synthetase

Nonribosomal peptide synthetases (NRPSs) are modular multidomain enzymes that act as an assembly line to catalyze the biosynthesis of complex natural products. The crystal structure of the 144-kilodalton Bacillus subtilis termination module SrfA-C was solved at 2.6 angstrom resolution. The adenylation and condensation domains of SrfA-C associate closely to form a catalytic platform, with their active sites on the same side of the platform. The peptidyl carrier protein domain is flexibly tethered to this platform and thus can move with its substrate-loaded 4'-phosphopantetheine arm between the active site of the adenylation domain and the donor side of the condensation domain. The SrfA-C crystal structure has implications for the rational redesign of NRPSs as a means of producing novel bioactive peptides.     

Dioxygen binds end-on to mononuclear copper in a precatalytic enzyme complex

Copper active sites play a major role in enzymatic activation of dioxygen. We trapped the copper-dioxygen complex in the enzyme peptidylglycine-alphahydroxylating monooxygenase (PHM) by freezing protein crystals that had been soaked with a slow substrate and ascorbate in the presence of oxygen. The x-ray crystal structure of this precatalytic complex, determined to 1.85-angstrom resolution, shows that oxygen binds to one of the coppers in the enzyme with an end-on geometry. Given this structure, it is likely that dioxygen is directly involved in the electron transfer and hydrogen abstraction steps of the PHM reaction. These insights may apply to other copper oxygen-activating enzymes, such as dopamine beta-monooxygenase, and to the design of biomimetic complexes.     

Structural evidence for a two-metal-ion mechanism of group I intron splicing

We report the 3.4 angstrom crystal structure of a catalytically active group I intron splicing intermediate containing the complete intron, both exons, the scissile phosphate, and all of the functional groups implicated in catalytic metal ion coordination, including the 2'-OH of the terminal guanosine. This structure suggests that, like protein phosphoryltransferases, an RNA phosphoryltransferase can use a two-metal-ion mechanism. Two Mg2+ ions are positioned 3.9 angstroms apart and are directly coordinated by all six of the biochemically predicted ligands. The evolutionary convergence of RNA and protein active sites on the same inorganic architecture highlights the intrinsic chemical capacity of the two-metal-ion catalytic mechanism for phosphoryl transfer.     

Electrostatic and steric contributions to regulation at the active site of isocitrate dehydrogenase

The isocitrate dehydrogenase of Escherichia coli is regulated by covalent modification at the active site rather than, as expected, at an allosteric site. As a means of evaluating the mechanism of regulation, the kinetics of the substrate, 2R,3S-isocitrate, and a substrate analog, 2R-malate, were compared for the native, phosphorylated, and mutant enzymes. Phosphorylation decreases activity by more than a factor of 10(6) for the true substrate, but causes minor changes in the activity of the substrate analog. The kinetic results indicate that electrostatic repulsion and steric hindrance between the phosphoryl moiety and the gamma carboxyl group of 2R,3S-isocitrate are the major causes of the inactivation, with a lesser contribution from the loss of a hydrogen bond.     

Active human-yeast chimeric phosphoglycerate kinases engineered by domain interchange

Phosphoglycerate kinase (PGK) is a monomeric protein composed of two domains of approximately equal size, connected by a hinge. Substrate-induced conformational change results in the closure of the active site cleft, which is situated between these two domains. In a study of the relations between structure and function of this enzyme, two interspecies hybrids were constructed, each composed of one domain from the human enzyme and one domain from the yeast enzyme. Despite a 35% difference in the amino acid composition between human and yeast PGK, catalytic properties of the hybrid enzymes are very similar to those of the parental proteins. This result demonstrates that the evolutionary substitutions within these two distantly related molecules do not significantly affect formation of the active site cleft, mechanism of domain closure, or enzyme activity itself.     

Structure of fungal fatty acid synthase and implications for iterative substrate shuttling

We report crystal structures of the 2.6-megadalton alpha6beta6 heterododecameric fatty acid synthase from Thermomyces lanuginosus at 3.1 angstrom resolution. The alpha and beta polypeptide chains form the six catalytic domains required for fatty acid synthesis and numerous expansion segments responsible for extensive intersubunit connections. Detailed views of all active sites provide insights into substrate specificities and catalytic mechanisms and reveal their unique characteristics, which are due to the integration into the multienzyme. The mode of acyl carrier protein attachment in the reaction chamber, together with the spatial distribution of active sites, suggests that iterative substrate shuttling is achieved by a relatively restricted circular motion of the carrier domain in the multifunctional enzyme.     

Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease

The rational design of drugs that can inhibit the action of viral proteases depends on obtaining accurate structures of these enzymes. The crystal structure of chemically synthesized HIV-1 protease has been determined at 2.8 angstrom resolution (R factor of 0.184) with the use of a model based on the Rous sarcoma virus protease structure. In this enzymatically active protein, the cysteines were replaced by alpha-amino-n-butyric acid, a nongenetically coded amino acid. This structure, in which all 99 amino acids were located, differs in several important details from that reported previously by others. The interface between the identical subunits forming the active protease dimer is composed of four well-ordered beta strands from both the amino and carboxyl termini and residues 86 to 94 have a helical conformation. The observed arrangement of the dimer interface suggests possible designs for dimerization inhibitors.     

类芦根细胞壁对铅的吸附固定机制

为探究类芦对Pb的耐性机制,采用对根细胞壁化学改性处理,测定Pb吸附动力学。结果表明,通过酯化改性去除部分羟基官能团后的根细胞壁对Pb~(2+)的吸附量相对降低了68.1%,其次为通过果胶酶改性去除部分果胶和通过甲基化改性去除部分氨基后的细胞壁,对Pb2+的吸附量相对降低48.9%与41.1%。利用傅里叶红外光谱分析,再结合FTIR图可得出,果胶提供的羟基基团、纤维素和半纤维素提供的羧基基团以及细胞壁蛋白提供的氨基官能团都是类芦固定Pb~(2+)的重要位点。研究表明,细胞壁组分中的羟基、羧基和氨基可以提供Pb结合位点,特别是羟基官能团可以使细胞壁对Pb具有较高的吸附固定能力,这也是类芦能够抵御Pb胁迫的一种重要机制。     

The complete primary structure of protein kinase C--the major phorbol ester receptor

Protein kinase C, the major phorbol ester receptor, was purified from bovine brain and through the use of oligonucleotide probes based on partial amino acid sequence, complementary DNA clones were derived from bovine brain complementary DNA libraries. Thus, the complete amino acid sequence of bovine protein kinase C was determined, revealing a domain structure. At the amino terminal is a cysteine-rich domain with an internal duplication; a putative calcium-binding domain follows, and there is at the carboxyl terminal a domain that shows substantial homology, but not identity, to sequences of other protein kinase.     

Architecture of a fungal fatty acid synthase at 5 A resolution

All steps of fatty acid synthesis in fungi are catalyzed by the fatty acid synthase, which forms a 2.6-megadalton alpha6beta6 complex. We have determined the molecular architecture of this multienzyme by fitting the structures of homologous enzymes that catalyze the individual steps of the reaction pathway into a 5 angstrom x-ray crystallographic electron density map. The huge assembly contains two separated reaction chambers, each equipped with three sets of active sites separated by distances up to approximately 130 angstroms, across which acyl carrier protein shuttles substrates during the reaction cycle. Regions of the electron density arising from well-defined structural features outside the catalytic domains separate the two reaction chambers and serve as a matrix in which domains carrying the various active sites are embedded. The structure rationalizes the compartmentalization of fatty acid synthesis, and the spatial arrangement of the active sites has specific implications for our understanding of the reaction cycle mechanism and of the architecture of multienzymes in general.     

基于细胞壁吸附固定特性的小飞蓬耐Cd机制研究

为探究小飞蓬根、叶细胞壁在Cd吸收过程中发挥的作用,以小飞蓬为供试植物,扫描电镜(SEM)观察Cd在小飞蓬组织中的分布及受损情况,通过细胞壁化学改性,利用吸附动力学和傅里叶红外光谱技术(FTIR)对小飞蓬根、叶细胞壁的Cd吸附固定特性进行了研究。扫描电镜结果显示,Cd胁迫下小飞蓬根、叶组织结构排列不规则,并出现晶体堵塞导管的现象,皮层组织是小飞蓬吸附固定Cd的重要部位;经酯化改性、氨基甲基化改性和果胶酶改性后,吸附动力学试验表明根细胞壁对Cd的累积吸附量分别相对降低了49.1%、38.5%和26.1%,叶细胞壁分别相对降低了39.47%、20.14%和30.23%。根细胞壁上的羧基和氨基在Cd吸附中的贡献作用较大,而叶细胞壁上的羧基和果胶在Cd吸附中的作用较明显。利用FTIR表征了小飞蓬根、叶细胞壁上Cd吸附位点的官能团信息后表明,在小飞蓬根、叶细胞壁吸附Cd的过程中,羟基、羧基和氨基是Cd的主要结合位点。其中,果胶为Cd的结合提供羟基官能团,纤维素和半纤维素为Cd的结合提供羧基官能团,而细胞壁蛋白提供了氨基官能团等结合位点。由此可见,细胞壁各组分中的羟基、羧基和氨基提供Cd结合位点,使细胞壁对Cd具有较高的吸附固定能力,是小飞蓬耐Cd的一种重要机制。     

Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.     

Direct observation of molecular preorganization for chirality transfer on a catalyst surface

The chemisorption of specific optically active compounds on metal surfaces can create catalytically active chirality transfer sites. However, the mechanism through which these sites bias the stereoselectivity of reactions (typically hydrogenations) is generally assumed to be so complex that continued progress in the area is uncertain. We show that the investigation of heterogeneous asymmetric induction with single-site resolution sufficient to distinguish stereochemical conformations at the submolecular level is finally accessible. A combination of scanning tunneling microscopy and density functional theory calculations reveals the stereodirecting forces governing preorganization into precise chiral modifier-substrate bimolecular surface complexes. The study shows that the chiral modifier induces prochiral switching on the surface and that different prochiral ratios prevail at different submolecular binding sites on the modifier at the reaction temperature.     

Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family

The three-dimensional structure of spinach ferredoxin-NADP+ reductase (NADP+, nicotinamide adenine dinucleotide phosphate) has been determined by x-ray diffraction at 2.6 angstroms (A) resolution and initially refined to an R factor of 0.226 at 2.2 A resolution. The model includes the flavin-adenine dinucleotide (FAD) prosthetic group and the protein chain from residue 19 through the carboxyl terminus at residue 314 and is composed of two domains. The FAD binding domain (residues 19 to 161) has an antiparallel beta barrel core and a single alpha helix for binding the pyrophosphate of FAD. The NADP binding domain (residues 162 to 314) has a central five-strand parallel beta sheet and six surrounding helices. Binding of the competitive inhibitor 2'-phospho-AMP (AMP, adenosine monophosphate) places the NADP binding site at the carboxyl-terminal edge of the sheet in a manner similar to the nucleotide binding of the dehydrogenase family. The structures reveal the key residues that function in cofactor binding and the catalytic center. With these key residues as a guide, conclusive evidence is presented that the ferredoxin reductase structure is a prototype for the nicotinamide dinucleotide and FAD binding domains of the enzymes NADPH-cytochrome P450 reductase, NADPH-sulfite reductase, NADH-cytochrome b5 reductase, and NADH-nitrate reductase. Thus this structure provides a structural framework for the NADH- or NADPH-dependent flavoenzyme parts of five distinct enzymes involved in photosynthesis, in the assimilation of inorganic nitrogen and sulfur, in fatty-acid oxidation, in the reduction of methemoglobin, and in the metabolism of many pesticides, drugs, and carcinogens.     

Crystal structure of Cd,Zn metallothionein

The anomalous scattering data from five Cd in the native protein were used to determine the crystal structure of cadmium, zinc (Cd,Zn) metallothionein isoform II from rat liver. The structure of a 4-Cd cluster was solved by direct methods. A 2.3 A resolution electron density map was calculated by iterative single-wavelength anomalous scattering. The structure is folded into two domains. The amino terminal domain (beta) of residues 1 to 29 enfolds a three-metal cluster of one Cd and two Zn atoms coordinated by six terminal cysteine thiolate ligands and three bridging cysteine thiolates. The carboxyl terminal domain (alpha) of residues 30 to 61 enfolds a 4-Cd cluster coordinated by six terminal and five bridging cysteine thiolates. All seven metal sites have tetrahedral coordination geometry. The domains are roughly spherical, and the diameter is 15 to 20 A; there is limited contact between domains. The folding of alpha and beta is topologically similar but with opposite chirality. Redundant, short cysteine-containing sequences have similar roles in cluster formation in both alpha and beta.     

Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.     

丝氨酸蛋白酶研究进展

综述了丝氨酸蛋白酶的结构与功能、蛋白质工程、表达系统和应用。丝氨酸蛋白酶是一类以丝氨酸为活性中心的重要的蛋白水解酶,在生物有机体中发挥着重要而又广泛的生理作用,具有广泛的研究和应用价值。它们的活性部位都含Ser、His、Asp,并具有相同的催化机制。丝氨酸蛋白酶超家族成员执行多种多样的生理功能,在很多致病过程以及细胞内的信号转导等方面起着重要作用。正是由于丝氨酸蛋白酶在结构上的微小变化,从而引起了其在功能上的进化。     

Autophosphorylation of protein kinase C at three separated regions of its primary sequence

The major autophosphorylation sites of the rat beta II isozyme of protein kinase C were identified. The modified threonine and serine residues were found in the amino-terminal peptide, the carboxyl-terminal tail, and the hinge region between the regulatory lipid-binding domain and the catalytic kinase domain. Because this autophosphorylation follows an intrapeptide mechanism, extraordinary flexibility of the protein is necessary to phosphorylate the three regions. Comparison of the sequences surrounding the modified residues showed no obvious recognition motif nor any similarity to substrate phosphorylation sites, suggesting that proximity to the active site may be the primary criterion for their phosphorylation.     

Structure of recombinant human renin, a target for cardiovascular-active drugs, at 2.5 A resolution

The x-ray crystal structure of recombinant human renin has been determined. Molecular dynamics techniques that included crystallographic data as a restraint were used to improve an initial model based on porcine pepsinogen. The present agreement factor for data from 8.0 to 2.5 angstroms (A) is 0.236. Some of the surface loops are poorly determined, and these disordered regions border a 30 A wide solvent channel. Comparison of renin with other aspartyl proteinases shows that, although the structural cores and active sites are highly conserved, surface residues, some of which are critical for specificity, vary greatly (up to 10A). Knowledge of the actual structure, as opposed to the use of models based on related enzymes, should facilitate the design of renin inhibitors.