Restricted Substrate Research Articles

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

One millimetre makes the difference: high-resolution analysis of methane-oxidizing bacteria and their specific activity at the oxic–anoxic interface in a flooded paddy soil

Aerobic methane-oxidizing bacteria (MOB) use a restricted substrate range, yet >30 species-equivalent operational taxonomical units (OTUs) are found in one paddy soil. How these OTUs physically share their microhabitat is unknown. Here we highly resolved the vertical distribution of MOB and their activity. Using microcosms and cryosectioning, we sub-sampled the top 3-mm of a water-saturated soil at near in situ conditions in 100-μm steps. We assessed the community structure and activity using the particulate methane monooxygenase gene pmoA as a functional and phylogenetic marker by terminal restriction fragment length polymorphism (t-RFLP), a pmoA-specific diagnostic microarray, and cloning and sequencing. pmoA genes and transcripts were quantified using competitive reverse transcriptase PCR combined with t-RFLP. Only a subset of the methanotroph community was active. Oxygen microprofiles showed that 89% of total respiration was confined to a 0.67-mm-thick zone immediately above the oxic-anoxic interface, most probably driven by methane oxidation. In this zone, a Methylobacter-affiliated OTU was highly active with up to 18 pmoA transcripts per cell and seemed to be adapted to oxygen and methane concentrations in the micromolar range. Analysis of transcripts with a pmoA-specific microarray found a Methylosarcina-affiliated OTU associated with the surface zone. High oxygen but only nanomolar methane concentrations at the surface suggested an adaptation of this OTU to oligotrophic conditions. No transcripts of type II methanotrophs (Methylosinus, Methylocystis) were found, which indicated that this group was represented by resting stages only. Hence, different OTUs within a single guild shared the same microenvironment and exploited different niches.

Restricted processing of glycans by endomannosidase in mammalian cells

Removal of α-glucose residues from nascent glycoproteins in the early secretory pathway is a requirement for further N-glycan maturation. Although deglucosylation is a stepwise process mediated by endoplasmic reticulum-associated glucosidases I and II for most glycoproteins, Golgi endo-α-mannosidase provides a backup mechanism for glycoprotein deglucosylation. Although conserved in mammals, in certain cell lines, endomannosidase activity in vitro appears to differ from its activity in cells following glucosidase inhibition. Here, we show that in bovine cells this is explained by restricted substrate specificity allowing processing of Glc(1)Man(7)GlcNAc(1/2) and Glc(1)Man(5)GlcNAc(1/2) but not fully glucosylated glycans that build up when glucosidases are inhibited. Our data further demonstrate that such specificity is determined genetically rather than post-translationally. We also demonstrate that the bovine endomannosidase is transcriptionally upregulated by comparison with glucosidase II in Madin-Darby bovine kidney cells and speculate that this is to compensate for the reduced catalytic activity as measured in the recombinant form of the enzyme.

Overexpression and oncogenic function of aldo-keto reductase family 1B10 (AKR1B10) in pancreatic carcinoma

AbstractAldo-keto reductase family 1B10 (AKR1B10) exhibits more restricted lipid substrate specificity (including farnesal, geranylgeranial, retinal and carbonyls), and metabolizing these lipid substrates has a crucial role in promoting carcinogenesis. Overexpression of AKR1B10 has been identified in smoking-related carcinomas such as lung cancer. As development of pancreatic cancer is firmly linked to smoking, the aim of the present study was to examine the expression and oncogenic role of AKR1B10 in pancreatic adenocarcinoma. AKR1B10 expression was analyzed in 50 paraffin-embedded clinical pancreatic cancer samples using immunohistochemistry. Oncogenic function of AKR1B10 was examined in pancreatic carcinoma cells in vitro using western blotting and siRNA approaches, mainly on cell apoptosis and protein prenylation including KRAS protein and its downstream signals. Immunohistochemistry analysis revealed that AKR1B10 overexpressed in 70% (35/50) of pancreatic adenocarcinomas and majority of pancreatic intraepithelial neoplasia, but not in adjacent morphologically normal pancreatic tissue. Compared with a normal pancreatic ductal epithelial cell (HPDE6E7), all of the six cultured pancreatic adenocarcinoma cell lines had an overexpression of AKR1B10 using immunoblotting, which correlated with increase of enzyme activity. siRNA-mediated silencing of AKR1B10 expression in pancreatic cancer cells resulted in (1) increased cell apoptosis, (2) increased non-farnesyled HDJ2 protein and (3) decreased membrane-bound prenylated KRAS protein and its downstream signaling molecules including phosphorylated ERK and MEK and membrane-bound E-cadherin. Our findings provide first time evidence that AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis possibly via modulation of cell apoptosis and protein prenylation.

Oleispira lenta sp. nov., a novel marine bacterium isolated from Yellow sea coastal seawater in Qingdao, China

The taxonomic position of strain DFH11(T), which was isolated from coastal seawater off Qingdao, People's Republic of China in 2007, was determined. Strain DFH11(T) comprised Gram-negative, motile, strictly aerobic spirilli that did not produce catalase. Comparative 16S rRNA gene sequence analysis revealed that strain DFH11(T) shared ~97.2, 93.3, 91.8, 91.7 and 91.5% sequence similarities with Oleispira antarctica, Spongiispira norvegica, Bermanella marisrubri, Oceaniserpentilla haliotis and Reinekea aestuarii, respectively. DNA-DNA hybridization experiments indicated that the strain was distinct from its closest phylogenetic neighbour, O. antarctica. The strain grew optimally in 2-3% (w/v) NaCl, at pH 5.0-10.0 (optimally at pH 7.0) and between 0 and 30°C (optimum growth temperature 28°C). The strain exhibited a restricted substrate profile, with a preference for aliphatic hydrocarbons, that is consistent with its closest phylogenetic neighbour O. antarctica. Growth of the isolate at different temperatures affected the cellular fatty acid profile. 28°C cultured cells contained C(16:1)ω7c and/or iso-C(15:0) 2-OH (50.4%) and C(16:0) (19.2%) as the major fatty acids. However, the major fatty acids of the cells cultured at 4°C were C(16:1)ω7c and/or C(16:1)ω6c (40.2%), C(16:0) (17.2%) and C(17:1)ω8c (10.1%). The G+C content of the genomic DNA was 42.7mol%. Phylogeny based on 16S rRNA gene sequences together with data from DNA-DNA hybridization, phenotypic and chemotaxonomic characterization revealed that DFH11(T) should be classified as a novel species of the genus Oleispira, for which the name Oleispira lenta sp. nov. is proposed, with the type strain DFH11(T) (=NCIMB 14529(T)=LMG 24829(T)).

The role of ADAM-mediated shedding in vascular biology

Within the vasculature the disintegrins and metalloproteinases (ADAMs) 8, 9, 10, 12, 15, 17, 19, 28 and 33 are expressed on endothelial cells, smooth muscle cells and on leukocytes. As surface-expressed proteases they mediate cleavage of vascular surface molecules at an extracellular site close to the membrane. This process is termed shedding and leads to the release of a soluble substrate ectodomain thereby critically modulating the biological function of the substrate. In the vasculature several surface molecules undergo ADAM-mediated shedding including tumour necrosis factor (TNF) α, interleukin (IL) 6 receptor α, L-selectin, vascular endothelial (VE)-cadherin, the transmembrane CX3C-chemokine ligand (CX3CL) 1, Notch, transforming growth factor (TGF) and heparin-binding epidermal growth factor (HB-EGF). These substrates play distinct roles in vascular biology by promoting inflammation, permeability changes, leukocyte recruitment, resolution of inflammation, regeneration and/or neovascularisation. Especially ADAM17 and ADAM10 are capable of cleaving many substrates with diverse function within the vasculature, whereas other ADAMs have a more restricted substrate range. Therefore, targeting ADAM17 or ADAM10 by pharmacologic inhibition or gene knockout not only attenuates the inflammatory response in animal models but also affects tissue regeneration and neovascularisation. Recent discoveries indicate that other ADAMs (e.g. ADAM8 and 9) also play important roles in vascular biology but appear to have more selective effects on vascular responses (e.g. on neovascularisation only). Although, targeting of ADAM17 and ADAM10 in inflammatory diseases is still a promising approach, temporal and spatial as well as substrate-specific inhibition approaches are required to minimise undesired side effects on vascular cells.

S1 Pocket of a Bacterially Derived Subtilisin-like Protease Underpins Effective Tissue Destruction

The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96% sequence identity. At present, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic but smaller than that of BprB. We show that BprV is more effective than BprB in degrading extracellular matrix components of the host tissue. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1 Leu-containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process.

Root colonisation by AM fungi differs between gypsum specialist and non-specialist plants: Links to the gypsophile behaviour

Gypsum soils are among the most restrictive substrates for plant life, yet the mechanisms of plant adaptation to gypsum are still poorly understood. Arbuscular mycorrhizal fungi (AMF) can improve host plant nutrition and survival in stressful environments but little is known about the ubiquity and function of AMF in plants that grow in gypsum soils, both specialists and generalists. Previous studies indicate that most gypsophiles (specialists) show much higher concentration of nutrients than gypsovags (generalists), hence our hypothesis was that this would be related to increased mycorrhizal colonisation in gypsum specialists. We therefore quantified colonisation of the roots by mycorrhizal arbuscules (AC), vesicles (VC) and hyphae (HC) in six species of gypsophiles and six species of gypsovags growing in gypsum outcrops. Both groups of plants showed significant differences in AC, VC and HC but in contrast to our hypothesis, colonisation was greater in gypsovags than in gypsophiles. The extent of AMF colonisation does not seem to explain the distinctively high nutrient concentrations reported for gypsophiles. Our results indicate that increased AM colonisation could be a mechanism allowing non-specialist plants to cope with the restrictive conditions of gypsum.

Biochemical characterisation of a NADPH-dependent carbonyl reductase from Neurospora crassa reducing α- and β-keto esters

A gene encoding an NADPH-dependent carbonyl reductase from Neurospora crassa ( nccr) was cloned and heterologously expressed in Escherichia coli. The enzyme (NcCR) was purified and biochemically characterised. NcCR exhibited a restricted substrate spectrum towards various ketones, and the highest activity (468 U/mg) was observed with dihydroxyacetone. However, NcCR proved to be very selective in the reduction of different α- and β-keto esters. Several compounds were converted to the corresponding hydroxy ester in high enantiomeric excess ( ee) at high conversion rates. The enantioselectivity of NcCR for the reduction of ethyl 4-chloro-3-oxobutanoate showed a strong dependence on temperature. This effect was studied in detail, revealing that the ee could be substantially increased by decreasing the temperature from 40 °C (78.8%) to −3 °C (98.0%). When the experimental conditions were optimised to improve the optical purity of the product, ( S)-4-chloro-3-hydroxybutanoate ( ee 98.0%) was successfully produced on a 300 mg (1.8 mmol) scale using NcCR at −3 °C.

Clinical Parameters Associated with Complete Cure of Atrial Fibrillation Undergoing Extensive Pulmonary Vein Isolation

Background: Little is known about clinical predictors for the recurrence after extensive pulmonary vein isolation (EPVI) in patients with atrial fibrillation (AF). Objectives: The purpose of this study was to investigate clinical parameters which predict complete cure of AF after EPVI. Methods and Results: A total of 152 consecutive patients with paroxysmal AF (PAF) who underwent successful EPVI were examined. We divided them into two groups, 1: Complete cure group; 103 patients who were free from PAF without any antiarrhythmic drugs and 2: recurrence group; the remaining 49 patients with recurrence of PAF. Between the two groups, we compared several clinical parameters before EPVI as follows; clinical backgrounds (presence of structural heart disease and CHADS2 score), parameters in transthoracic and transesophageal echocardiogram [left atrium (LA) dimension, systolic and diastolic LA appendage (LAA) flow velocity], LA volume assessed by multiditector CT, additional creation of cava-tricuspid isthmus block line, serum brain natriuretic peptide (BNP) level. Using multi-variate analysis, filling velocity of the LAA and BNP before EPVI were identified as the independent predictors of complete cure of PAF (p<0.001, p<0.05, respectively). Conclusion: Low BNP levels and better filling velocity of the LAA are the negative predictors for the recurrence after EPVI in patients with AF. These may relate to preserved LA function and to restricted AF substrates within PVs.

Structure and Function of APH(4)-Ia, a Hygromycin B Resistance Enzyme

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.

Mechanisms of factor VIIa-catalyzed activation of factor VIII.

Factor (F)VIIa, complexed with tissue factor (TF), is a primary trigger of blood coagulation, and has extremely restricted substrate specificity. The complex catalyzes limited proteolysis of FVIII, but these mechanisms are poorly understood. In the present study, we investigated the precise mechanisms of FVIIa/TF-catalyzed FVIII activation. FVIII activity increased ~4-fold within 30 s in the presence of FVIIa/TF, and then decreased to initial levels within 20 min. FVIIa (0.1 nM), at concentrations present physiologically in plasma, activated FVIII in the presence of TF, and this activation was more rapid than that induced by thrombin. The heavy chain (HCh) of FVIII was proteolyzed at Arg(740) and Arg(372) more rapidly by FVIIa/TF than by thrombin, consistent with the enhanced activation of FVIII. Cleavage at Arg(336) was evident at ~1 min, whilst little cleavage of the light chain (LCh) was observed. Cleavage of the HCh by FVIIa/TF was governed by the presence of the LCh. FVIII bound to Glu-Gly-Arg-active-site-modified FVIIa (K(d), ~0.8 nM) with a higher affinity for the HCh than for the LCh (K(d), 5.9 and 18.9 nm). Binding to the A2 domain was particularly evident. Von Willebrand factor (VWF) modestly inhibited FVIIa/TF-catalyzed FVIII activation, in keeping with the concept that VWF could moderate FVIIa/TF-mediated reactions. The results demonstrated that this activation mechanism was distinct from those mediated by thrombin, and indicated that FVIIa/TF functions through a 'priming' mechanism for the activation of FVIII in the initiation phase of coagulation.

Intramolecular Hydroamination/Cyclization of Aminoalkenes Catalyzed by Ln[N(SiMe3)2]3 Grafted onto Periodic Mesoporous Silicas

Homoleptic rare-earth metal silylamide complexes Ln[N(SiMe(3))(2)](3) (Ln = Y, La, Nd) were grafted onto a series of partially dehydroxylated periodic mesoporous silica (PMS) supports, SBA-15(-500) (d(p) = 7.9 nm), SBA-15LP(-500) (d(p) = 16.6 nm), and MCM-41(-500) (d(p) = 4.1 nm). The hybrid materials Ln[N(SiMe(3))(2)](3)@PMS efficiently catalyze the intramolecular hydroamination/cyclization reaction of 2,2-dimethyl-4-penten-1-amine. Under the prevailing slurry conditions the metal size (Y > La > Nd), the pore size, and the particle morphology affect the catalytic performance. Material Y[N(SiMe(3))(2)](3)@SBA-15LP(-500) displayed the highest activity (TOF = up to 420 h(-1) at 60 °C), with the extralarge pores minimizing restrictive product inhibition and substrate diffusion effects. The catalytic activity of Y[N(SiMe(3))(2)](3)@SBA-15LP(-500) is found to be much higher than that of the molecular counterpart (TOF = up to 54 h(-1)), and its recyclability is demonstrated.

A Novel Glycerophosphodiester Phosphodiesterase, GDE5, Controls Skeletal Muscle Development via a Non-enzymatic Mechanism

Mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5DeltaC471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5DeltaC471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism.

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the X-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the X-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.

The reductase domain in a Type I fatty acid synthase from the apicomplexan Cryptosporidium parvum: Restricted substrate preference towards very long chain fatty acyl thioesters

BackgroundThe apicomplexan Cryptosporidium parvum genome possesses a 25-kb intronless open reading frame (ORF) that predicts a multifunctional Type I fatty acid synthase (CpFAS1) with at least 21 enzymatic domains. Although the architecture of CpFAS1 resembles those of bacterial polyketide synthases (PKSs), this megasynthase is predicted to function as a fatty acyl elongase as our earlier studies have indicated that the N-terminal loading unit (acyl-[ACP] ligase) prefers using intermediate to long chain fatty acids as substrates, and each of the three internal elongation modules contains a complete set of enzymes to produce a saturated fatty acyl chain. Although the activities of almost all domains were confirmed using recombinant proteins, that of the C-terminal reductase domain (CpFAS1-R) was yet undetermined. In fact, there were no published studies to report the kinetic features of any reductase domains in bacterial PKSs using purified recombinant or native proteins.ResultsIn the present study, the identity of CpFAS1-R as a reductase is confirmed by in silico analysis on sequence similarity and characteristic motifs. Phylogenetic analysis based on the R-domains supports a previous notion on the bacterial origin of apicomplexan Type I FAS/PKS genes. We also developed a novel assay using fatty acyl-CoAs as substrates, and determined that CpFAS1-R could only utilize very long chain fatty acyl-CoAs as substrates (i.e., with activity on C26 > C24 > C22 > C20, but no activity on C18 and C16). It was capable of using both NADPH and NADH as electron donors, but prefers NADPH to NADH. The activity of CpFAS1-R displayed allosteric kinetics towards C26 hexacosanoyl CoA as a substrate (h = 2.0; Vmax = 32.8 nmol min-1 mg-1 protein; and K50 = 0.91 mM).ConclusionsWe have confirmed the activity of CpFAS1-R by directly assaying its substrate preference and kinetic parameters, which is for the first time for a Type I FAS, PKS or non-ribosomal peptide synthase (NRPS) reductase domain. The restricted substrate preference towards very long chain fatty acyl thioesters may be an important feature for this megasynthase to avoid the release of product(s) with undesired lengths.

Identification of substrates for cyclin dependent kinases

Protein phosphorylation is a key regulatory mechanism for cell cycle control in eukaryotes. From yeast to humans, cell cycle progression and cell division require the activation of a group of serine– threonine protein kinases called cyclin dependent kinases (CDKs) (Morgan, 1997), which initiate and coordinate these processes by orderly phosphorylation of their targets. CDKs require association with a cyclin subunit to activate them,whichprovides substrate specificity (Russo,1997; Solomonet al.,1992), and phosphorylation by an activating protein kinase (CAK) at a conserved threonine residue (Krek and Nigg,1992; Lorca et al., 1992; Solomon et al., 1992). The concentration of cyclins oscillate during the cell cycle and their abundance is regulated at several levels: transcriptional, translational and at the level of protein stability. In particular, at the end of mitosis, ubiquitin-mediated proteolysis results in the disappearance of mitotic cyclins, which results in inactivation of CDKs (Zachariae and Nasmyth, 1999). The key cell cycle transitions involving the activity of CDKs occur at the initiation of S-phase and entry into mitosis. Loss of CDK activity at the end of mitosis permits the return to interphase, and is presumably accompanied by dephosphorylation of the majority of target proteins that were modified when the cells entered mitosis. There is rather specific control of the different phases of the cell cycle, so that cells do not enter mitosis with unreplicated or damaged DNA, and do not exit mitosis until all the chromosomes are correctly aligned on the metaphase plate. There is a theoretical conundrum, however, in explaining how it is that activation of S-phase CDKs, such as cyclin E/CDK2 and cyclin A/ CDK2 in vertebrate cells promotes entry into S-phase and not into mitosis, or why activation of the Mphase CDKs does not promote a further round of DNA replication. One hypothesis to explain why cells enter S-phase, rather than mitosis, when the S-phase CDK (CDK2-cyclin E in higher eukaryotes) is activated would be a restricted substrate specificity for this particular CDK, yet there is very little information on this point. Another possibility is that CDK2-cyclin E resides in the nucleus, and entry

Comparison of Staphopain A (ScpA) and B (SspB) precursor activation mechanisms reveals unique secretion kinetics of proSspB (Staphopain B), and a different interaction with its cognate Staphostatin, SspC

The scpAB and sspABC operons of Staphylococcus aureus encode Staphopain cysteine proteases ScpA and SspB, and their respective Staphostatins ScpB and SspC, which are thought to protect against premature activation of Staphopain precursors during protein export. However, we found that the proSspB precursor was secreted and activated without detriment to S. aureus in the absence of SspC function. Our data indicate that this is feasible due to a restricted substrate specificity of mature SspB, a stable precursor structure and slow secretion kinetics. In contrast, mature ScpA had a broad substrate specificity, such that it was prone to autolytic degradation, but also was uniquely able to degrade elastin fibres. Modelling of proScpA relative to the proSspB structure identified several differences, which appear to optimize proScpA for autocatalytic activation, whereas proSspB is optimized for stability, and cannot initiate autocatalytic activation. Consequently, recombinant proSspB remained stable and unprocessed when retained in the cytoplasm of Escherichia coli, whereas proScpA initiated rapid autocatalytic activation, leading to capture of an activation intermediate by ScpB. We conclude that the status of sspBC in S. aureus, as paralogues of the ancestral scpAB genes, facilitated a different activation mechanism, a stable proSspB isoform and modified Staphostatin function.

Characterization of the Human Sulfatase Sulf1 and Its High Affinity Heparin/Heparan Sulfate Interaction Domain

The extracellular sulfatases Sulf1 and Sulf2 remodel the 6O-sulfation state of heparan sulfate proteoglycans on the cell surface, thereby modulating growth factor signaling. Different from all other sulfatases, the Sulfs contain a unique, positively charged hydrophilic domain (HD) of about 320 amino acid residues. Using various HD deletion mutants and glutathione S-transferase (GST)-HD fusion proteins, this study demonstrates that the HD is required for enzymatic activity and acts as a high affinity heparin/heparan sulfate interaction domain. Association of the HD with the cell surface is sensitive to heparinase treatment, underlining specificity toward heparan sulfate chains. Correspondingly, isolated GST-HD binds strongly to both heparin and heparan sulfate in vitro and also to living cells. Surface plasmon resonance studies indicate nanomolar affinity of GST-HD toward immobilized heparin. The comparison of different mutants reveals that especially the outer regions of the HD mediate heparan sulfate binding, probably involving "tandem" interactions. Interestingly, binding to heparan sulfate depends on the presence of 6O-sulfate substrate groups, suggesting that substrate turnover facilitates release of the enzyme from its substrate. Deletion of the inner, less conserved region of the HD drastically increases Sulf1 secretion without affecting enzymatic activity or substrate specificity, thus providing a tool for the in vitro modulation of HS-dependent signaling as demonstrated here for the signal transduction of fibroblast growth factor 2. Taken together, the present study shows that specific regions of the HD influence different aspects of HS binding, cellular localization, and enzyme function.

Identification of the Determinant Conferring Permissive Substrate Usage in the Telomere Resolvase, ResT

Linear genome stability requires specialized telomere replication and protection mechanisms. A common solution to this problem in non-eukaryotes is the formation of hairpin telomeres by telomere resolvases (also known as protelomerases). These enzymes perform a two-step transesterification on replication intermediates to generate hairpin telomeres using an active site similar to that of tyrosine recombinases and type IB topoisomerases. Unlike phage telomere resolvases, the telomere resolvase from the Lyme disease pathogen Borrelia burgdorferi (ResT) is a permissive enzyme that resolves several types of telomere in vitro. However, the ResT region and residues mediating permissive substrate usage have not been identified. The relapsing fever Borrelia hermsii ResT exhibits a more restricted substrate usage pattern than B. burgdorferi ResT and cannot efficiently resolve a Type 2 telomere. In this study, we determined that all relapsing fever ResTs process Type 2 telomeres inefficiently. Using a library of chimeric and mutant B. hermsii/B. burgdorferi ResTs, we mapped the determinants in B. burgdorferi ResT conferring the ability to resolve multiple Type 2 telomeres. Type 2 telomere resolution was dependent on a single proline in the ResT catalytic region that was conserved in all Lyme disease but not relapsing fever ResTs and that is part of a 2-amino acid insertion absent from phage telomere resolvase sequences. The identification of a permissive substrate usage determinant explains the ability of B. burgdorferi ResT to process the 19 unique telomeres found in its segmented genome and will aid further studies on the structure and function of this essential enzyme.