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Rationale: Defects in muscle glucose metabolism are linked to type 2 diabetes. Mechanistic studies examining these defects rely on the use of high fat-fed rodent models and typically involve the determination of muscle glucose uptake under insulin-stimulated conditions. While insightful, they do not necessarily reflect the physiology of the postprandial state. In addition, most studies do not examine aspects of glucose metabolism beyond the uptake process. Here we present an approach to study rodent muscle glucose and intermediary metabolism under the dynamic and physiologically relevant setting of the oral glucose tolerance test (OGTT)., Methods and Results: In vivo muscle glucose and intermediary metabolism was investigated following oral administration of [U-(13)C] glucose. Quadriceps muscles were collected 15 and 60 min after glucose administration and metabolite flux profiling was determined by measuring (13)C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates via gas chromatography-mass spectrometry. While no dietary effects were noted in the glycolytic pathway, muscle from mice fed a high fat diet (HFD) exhibited a reduction in labelling in TCA intermediates. Interestingly, this appeared to be independent of alterations in flux through pyruvate dehydrogenase. In addition, our findings suggest that TCA cycle anaplerosis is negligible in muscle during an OGTT., Conclusions: Under the dynamic physiologically relevant conditions of the OGTT, skeletal muscle from HFD fed mice exhibits alterations in glucose metabolism at the level of the TCA cycle., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The mammalian target of rapamycin (mTOR) pathway is the primary regulator of muscle protein synthesis. Metabolic syndrome (MetS) is characterized by central obesity and insulin resistance; little is known about how MetS affects the sensitivity of the mTOR pathway to feeding., Methods: The responsiveness of mTOR pathway targets such as p706Sk to a high protein meal containing either dairy or soy foods was investigated in healthy insulin sensitive middle-aged men and those presenting with metabolic syndrome (MetS). Twenty male subjects (10 healthy controls, 10 MetS) participated in a single-blinded randomized cross-over study. In a random sequence, subjects ingested energy-matched breakfasts composed predominately of either dairy-protein or soy-protein foods. Skeletal muscle biopsies were collected in the fasted state and at 2 and 4 h post-meal ingestion for the analysis of mTOR- and insulin-signalling kinase activation., Results: Phosphorylated Akt and Insulin receptor substrate 1 (IRS1) increased during the postabsorptive period with no difference between groups. mTOR (Ser448) and ribosomal protein S6 phosphorylation increased 2 h following dairy meal consumption only. p70S6K (Thr389) phosphorylation was increased after feeding only in the control subjects and not in the MetS group., Conclusions: These data demonstrate that the consumption of a dairy-protein rich but not a soy-protein rich breakfast activates the phosphorylation of mTOR and ribosomal protein S6, required for protein synthesis in human skeletal muscle. Unlike healthy controls, subjects with MetS did not increase muscle p70S6K(Thr389) phosphorylation in response to a mixed meal., Trial Registration: This trial was registered with the Australian New Zealand Clinical Trials Registry (ANZCTR) as ACTRN12610000562077.

Leishmania parasites express three highly conserved small myristoylated proteins (SMPs) that are targeted to distinct membranes. SMP-1 is exclusively found in the flagellum, depending on myristoylation and palmitoylation. In contrast, monoacylated SMP-2 and SMP-4 are localized to the flagellar pocket and plasma membrane, respectively. Here, we demonstrate that unlike SMP-4, SMP-2 resides in detergent resistant membranes, but can be readily solubilized in the presence of high concentrations of salt. We provide evidence that in detergent resistant membranes, SMP-2 forms high molecular weight complexes in vivo. Association with detergent resistant membranes was abrogated in the presence of a C-terminal tag suggesting acylation independent targeting signals. In addition, the N-terminal region of SMP-2 contains sufficient information for membrane targeting, as a GFP-chimera localizes to the flagellar pocket. Thus while the core sequences of the SMPs are highly conserved, individual members have evolved different mechanisms for their diverse membrane localization., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Metabolomics research often requires the use of multiple analytical platforms, batches of samples, and laboratories, any of which can introduce a component of unwanted variation. In addition, every experiment is subject to within-platform and other experimental variation, which often includes unwanted biological variation. Such variation must be removed in order to focus on the biological information of interest. We present a broadly applicable method for the removal of unwanted variation arising from various sources for the identification of differentially abundant metabolites and, hence, for the systematic integration of data on the same quantities from different sources. We illustrate the versatility and the performance of the approach in four applications, and we show that it has several advantages over the existing normalization methods.

Wastewater samples were taken from an aerobic bioreactor, operated by a dairy processor in southeastern Australia to reduce nutrient and pollutant loads. Samples were taken over a two-year period, to determine whether trace organic compounds or physicochemical analyses of the wastewater could be used to discriminate the water taken before, during and after processing of the wastewater in the bioreactor. Multivariate analyses of the physicochemical data suggested that nitrate, pH and total dissolved nitrogen best described the infeed wastewater entering the bioreactor, while organic and particulate phosphorus concentrations where predominantly responsible for describing the composition of the content of the bioreactor. Gas chromatography-mass spectrometry data of organic compounds within the wastewater samples were also analysed via multivariate analyses. The analyses found that the compound 4-nitrophenol was associated with ammonia concentrations and mixed liquor wastewater. Therefore, 4-nitrophenol may possibly be used to act as an indicator of anaerobicity in aerobic bioreactors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Trypanosomatid parasites express a number of mono- and diacylated proteins that are targeted to distinct regions of the plasma membrane including the cell body, the flagellum and the flagellar pocket. The extent to which the acylation status and other protein motifs regulate the targeting and/or retention of these proteins to the distinct membrane domains is poorly defined. We have previously described a family of small myristoylated proteins (SMPs) that are either monoacylated (myristoylated) or diacylated (myristoylated and palmitoylated) and targeted to distinct plasma membrane domains. Diacylated SMP-1 is a major constituent of the flagellar membrane, whereas monoacylated SMP-2 resides in the flagellar pocket in Leishmania major. Here, we show that a third SMP family member, monoacylated SMP-4, localizes predominantly to the pellicular membrane. Density gradient centrifugation of detergent-insoluble membranes indicated that SMP-4 was associated with detergent-insoluble domains but was not tightly associated with the subpellicular cytoskeleton. Based on the localisation of truncated SMP proteins, we conclude that the flagellum targeting of SMP-1 is primarily dependent on the dual-acylation motif. In contrast, the localisation of SMP-4 to the cell body membrane is dependent on N-terminal myristoylation and a C-terminal peptide subdomain with a predicted α-helical structure. Strikingly, a SMP-1 chimera containing the SMP-4 C-terminal extension was selectively trafficked to the distal tip of the flagellum and failed to complement the loss of native SMP-1 in a Δsmp1/2 double knockout strain. Collectively, these results suggest that dual acylation is sufficient to target some SMP proteins to the flagellum, while the unique C-terminal extensions of these proteins may confer additional membrane targeting signals that are important for both localisation and SMP function., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Leishmania parasites experience variable nutrient levels as they cycle between the extracellular promastigote stage in the sandfly vector and the obligate intracellular amastigote stage in the mammalian host. Here we show that the surface expression of three Leishmania mexicana hexose and myo-inositol transporters is regulated in both a stage-specific and nutrient-dependent manner. GFP-chimeras of functionally active hexose transporters, LmGT2 and LmGT3, and the myo-inositol transporter, MIT, were primarily expressed in the cell body plasma membrane in rapidly dividing promastigote stages. However MIT-GFP was mostly rerouted to the multivesicular tubule (MVT)-lysosome when promastigotes reached stationary phase growth and all three nutrient transporters were targeted to the amastigote lysosome following transformation to in vitro differentiated or in vivo imaged amastigote stages. This stage-specific decrease in surface expression of GFP-tagged transporters correlated with decreased hexose or myo-inositol uptake in stationary phase promastigotes and amastigotes. The MVT-lysosme targeting of the MIT-GFP protein was reversed when promastigotes were deprived of myo-inositol, indicating that nutrient signals can override stage-specific changes in transporter distribution. The surface expression of the hexose and myo-inositol transporters was not regulated by interactions with the subpellicular cytoskeleton, as both classes of transporters associated with detergent-resistant membranes. LmGT3-GFP and MIT-GFP proteins C-terminally modified with mono-ubiquitin were constitutively transported to the MVT-lysosome, suggesting that ubiquitination may play a key role in regulating the subcellular distribution of these transporters and parasite adaptation to different nutrient conditions., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Eukaryotic flagella and cilia are surrounded by a membrane that is continuous with, but distinct from, the rest of the plasma membrane. In Leishmania parasites, the inner leaflet of the flagellar membrane is coated with the acylated membrane protein, SMP-1. Here, we provide evidence that SMP-1 stabilizes the flagellar membrane and is required for flagella elongation and function. The expression and flagella targeting of SMP-1 is tightly associated with flagella elongation during amastigote to promastigote differentiation. Deletion of the genes encoding SMP-1 and the flagellar pocket protein SMP-2, led to the production of short flagella and defects in motility. Alterations in the physical properties of the smp-1/smp-2(-/-) flagellar membrane were suggested by: (1) the accumulation of membrane vesicles in the flagellar matrix, and (2) further retraction of flagella following partial inhibition of sterol and sphingolipid biosynthesis. The flagella phenotype of the smp-1/smp-2(-/-) null mutant was reversed by re-expression of SMP-1, but not SMP-2. SMP-1 contains a jelly-roll beta-sheet structure that is probably conserved in all SMP proteins, and forms stable homo-oligomers in vivo. We propose that the SMP-1 coat generates and/or stabilizes sterol- and sphingolipid-rich domains in the flagellar membrane.

The cell walls of the Corynebacterineae, which includes the important human pathogen Mycobacterium tuberculosis, contain two major lipopolysaccharides, lipoarabinomannan (LAM) and lipomannan (LM). LAM is assembled on a subpool of phosphatidylinositol mannosides (PIMs), whereas the identity of the LM lipid anchor is less well characterized. In this study we have identified a new gene (Rv2188c in M. tuberculosis and NCgl2106 in Corynebacterium glutamicum) that encodes a mannosyltransferase involved in the synthesis of the early dimannosylated PIM species, acyl-PIM2, and LAM. Disruption of the C. glutamicum NCgl2106 gene resulted in loss of synthesis of AcPIM2 and accumulation of the monomannosylated precursor, AcPIM1. The synthesis of a structurally unrelated mannolipid, Gl-X, was unaffected. The synthesis of AcPIM2 in C. glutamicum DeltaNCgl2106 was restored by complementation with M. tuberculosis Rv2188c. In vivo labeling of the mutant with [3H]Man and in vitro labeling of membranes with GDP-[3H]Man confirmed that NCgl2106/Rv2188c catalyzed the second mannose addition in PIM biosynthesis, a function previously ascribed to PimB/Rv0557. The C. glutamicum Delta NCgl2106 mutant lacked mature LAM but unexpectedly still synthesized the major pool of LM. Biochemical analyses of the LM core indicated that this lipopolysaccharide was assembled on Gl-X. These data suggest that NCgl2106/Rv2188c and the previously studied PimB/Rv0557 transfer mannose residues to distinct mannoglycolipids that act as precursors for LAM and LM, respectively.

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055DeltalpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055DeltalpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.

T cell differentiation in the thymus is dependent upon signals from thymic stromal cells. Most studies into the nature of these signals have focused only on the support provided by the thymic epithelium, but there is an emerging view that other stromal cells such as mesenchymal fibroblasts may also be involved. Study of the latter has been hindered by a lack of appropriate markers, particularly those allowing their isolation. In this study, we describe a new surface marker of thymic stroma, MTS-15, and demonstrate its specificity for fibroblasts and a subset of endothelial cells. Coculture experiments showed that the determinant could be transferred between cells. Extensive biochemical analysis demonstrated that the Ag bound by MTS-15 was the glycosphingolipid Forssman determinant, consistent with the distribution observed. Transcriptional analysis of purified MTS-15(+) thymic fibroblasts revealed a unique expression profile for a number of chemokines and growth factors important to thymocyte and epithelial cell development. In a model of cyclophosphamide-induced thymic involution and regeneration, fibroblasts were found to expand extensively and express growth factors important to epithelial proliferation and increased T cell production just before thymic regeneration. Overall, this study identifies a useful marker of thymic fibroblasts and highlights this subpopulation as a key player in thymic function by virtue of their support of both thymocytes and epithelial cells.

Mycolic acids are essential components of the cell walls of bacteria belonging to the suborder Corynebacterineae, including the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. Mycolic acid biosynthesis is complex and the target of several frontline antimycobacterial drugs. The condensation of two fatty acids to form a 2-alkyl-3-keto mycolate precursor and the subsequent reduction of this precursor represent two key and highly conserved steps in this pathway. Although the enzyme catalyzing the condensation step has recently been identified, little is known about the putative reductase. Using an extensive bioinformatic comparison of the genomes of M. tuberculosis and Corynebacterium glutamicum, we identified NCgl2385, the orthologue of Rv2509 in M. tuberculosis, as a potential reductase candidate. Deletion of the gene in C. glutamicum resulted in a slow growing strain that was deficient in arabinogalactan-linked mycolates and synthesized abnormal forms of the mycolate-containing glycolipids trehalose dicorynomycolate and trehalose monocorynomycolate. Analysis of the native and acetylated trehalose glycolipids by MALDI-TOF mass spectrometry indicated that these novel glycolipids contained an unreduced beta-keto ester. This was confirmed by analysis of sodium borodeuteride-reduced mycolic acids by gas chromatography mass spectrometry. Reintroduction of the NCgl2385 gene into the mutant restored the transfer of mature mycolic acids to both the trehalose glycolipids and cell wall arabinogalactan. These data indicate that NCgl2385, which we have designated CmrA, is essential for the production of mature trehalose mycolates and subsequent covalent attachment of mycolic acids onto the cell wall, thus representing a focus for future structural and pathogenicity studies.

The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PMf, that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PMf and PM-CW fractions. The PMf was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PMf marker enzymes to non-PM-CW fractions. The formation of the PMf and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.

The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.

The relative specificity and bond cleavage pattern of barley alpha-amylase 1 (AMY1) were dramatically changed by mutation in F(286)VD that connected beta-strand 7 of the catalytic (beta/alpha)(8)-barrel to a succeeding 3(10)-helix. This conserved tripeptide of the otherwise variable beta --> alpha segment 7 lacked direct ligand contact, but the nearby residues His290 and Asp291 participated in transition-state stabilization and catalysis. On the basis of sequences of glycoside hydrolase family 13, a biased random mutagenesis protocol was designed which encoded 174 putative F(286)VD variants of C95A-AMY1, chosen as the parent enzyme to avoid inactivating glutathionylation by the yeast host. The FVG, FGG, YVD, LLD, and FLE mutants showed 12-380 and 1.8-33% catalytic efficiency (k(cat)/K(m)) toward 2-chloro-4-nitrophenyl beta-D-maltoheptaoside and amylose DP17, respectively, and 0.5-50% activity for insoluble starch compared to that of C95A-AMY1. K(m) and k(cat) were decreased 2-9- and 1.3-83-fold, respectively, for the soluble substrates. The starch:oligosaccharide and amylose:oligosaccharide specificity ratios were 13-172 and 2.4-14 for mutants and 520 and 27 for C95A-AMY1, respectively. The FVG mutant released 4-nitrophenyl alpha-D-maltotrioside (PNPG(3)) from PNPG(5), whereas C95A-AMY1 produced PNPG and PNPG(2). The mutation thus favored interaction with the substrate aglycon part, while products from PNPG(6) reflected the fact that the mutation restored binding at subsite -6 which was lost in C95A-AMY1. The outcome of this combined irrational and rational protein engineering approach was evaluated considering structural accommodation of mutant side chains. FVG and FGG, present in the most active variants, represented novel sequences. This emphasized the worth of random mutagenesis and launched flexibility as a goal for beta --> alpha loop 7 engineering in family 13.

Alkalophilic Bacillus alpha-amylase (ABA) was produced in the yeast Pichia pastoris with a yield of 50 mg L(-1) of culture supernatant. The recombinant protein, rABA, was glycosylated at seven of the nine sites for potential N-glycosylation as identified by automated peptide sequencing and MALDI-TOF MS of tryptic fragments. The number of hexose units within each glycan chain was found to vary from 8 to 18 as calculated from the masses of glycosylated peptide fragments. Temperature stability measurements in the absence of substrate showed that the T(50) of glycosylated rABA and its endoglycosidase H-deglycosylated form was 76 degrees C while that of ABA purified from Bacillus was 89 degrees C thus demonstrating that the original temperature stability of ABA was not retained by rABA. The relative thermoperformance, i.e., the activity at 80 degrees C relative to that at 37 degrees C was 0.9 +/- 0.3 for rABA. Removal of all seven N-linked glycans by endoglycosidase H increased the relative thermoperformance to 2.4 +/- 0.6, compared to the value of 3.5 +/- 1.1 for ABA. Thus, removal of the N-linked glycans did not improve the thermostability of rABA but modified its thermoperformance to approach that of the original Bacillus enzyme. rABA had the highest activity around pH 6. Treatment of rABA with endoglycosidase H shifted the pH activity profile in a more alkaline direction approaching the pH activity profile of ABA., (Copyright 2001 Academic Press.)

The pKa of the catalytic base was lowered and its distance to the general acid catalyst, Glu179, was increased in the glucoamylase from Aspergillus awamori by replacing the catalytic base Glu400 with cysteine followed by oxidation to cysteinesulfinic acid [Fierobe, H.-P., Mirgorodskaya, E., McGuire, K. A., Roepstorff, P., Svensson, B. and Clarke, A. J. (1998) Biochemistry 37, 3743-3752. 1H NMR spectroscopy demonstrated that the oxidized mutant Glu400-->Cys-SO2H glucoamylase, like the wild-type, catalyzed hydrolysis with inversion of the anomeric configuration of the product. Relative to the catalytic base mutant Glu400-->Cys, the Cys400-SO2H glucoamylase had 700 times higher kcat toward maltose, while K(m) was unchanged. Compared to wild-type glucoamylase, the Cys400-SO2H derivative had kcat values of 150-190% and 85-320% on malto- and isomaltooligosaccharides, respectively, while K(m) values were similar to those of wild-type with the two disaccharides and 3.5-5.5- and 1.8-2.5-fold higher for the longer malto- and isomaltooligosaccharides substrates, respectively. The pH-activity dependence at saturating concentration of maltose indicated that the pKa of the catalytic base Cys400-SO2H was about 0.5 pH unit lower than that of wild-type Glu400. The Ki of Cys400-SO2H glucoamylase for the pseudotetrasaccharide and potent inhibitor acarbose increased more than 10(4)-fold, but Ki values of the mono- and disaccharide analogues 1-deoxynojirimycin and beta-O-methylacarviosinide were unchanged, suggesting perturbation at binding subsites beyond the catalytic center. A distinct property of Cys400-SO2H glucoamylase was the catalysis of the condensation of beta-D-glucopyranosyl fluoride and subsequent hydrolysis of the product to beta-glucose, under conditions where this was not detected for the wild-type enzyme.

The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions. Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue. However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the alpha-glycosyl azide. Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold. Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center. Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (> 10(5)-fold) effects on substrates with poor leaving groups. The Brønsted beta 1g value for hydrolysis of aryl cellobiosides was much larger (beta 1g approximately -1) for the mutant than for the wild-type enzyme (beta 1g = -0.3), consistent with removal of protonic assistance. The pH-dependence was also significantly perturbed. Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products. These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling.

Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae. The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2. Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose. The N-terminal sequence, pI, and Mr indicated that native-like processing took place. Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1. While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS. The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity. Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts. This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships.

We have identified the residue in Cellulomonas fimi exoglycanase modified by N-bromoacetyl cellobiosylamine as Glu 127 using a new combination of experimental approaches. The enzyme was quantitatively inhibited with the affinity label N-bromoacetyl cellobiosylamine and cleaved with pepsin. The N-acetyl cellobiosylamine-modified peptide was identified by comparative peptide mapping of the digests derived from labeled and unlabeled proteins by reverse-phase high-performance liquid chromatography connected online to an electrospray ionization mass spectrometer. The modified residue in the labeled peptide was determined by using a novel protein sequencing chemistry which is based on monitoring the amino acid derivatives released by stepwise peptide degradation using electrospray ionization mass spectrometry. Tandem mass spectrometry was used for further structural characterization of the cleaved residue. We show that the residue modified by N-bromoacetyl cellobiosylamine is Glu 127. This residue has been identified previously as the acid-base catalyst by using a combination of mutagenic and kinetic analyses. Our results therefore demonstrate the usefulness of this type of affinity label in identifying important catalytic residues in glycosidases and suggest that this new experimental approach can be applied generally to any labeled protein in which the mass of the label is known and thus represents an alternative approach to the current methods used to identify labeled residues within proteins.

The three-dimensional structure of a catalytically competent glycosyl-enzyme intermediate of a retaining beta-1,4-glycanase has been determined at a resolution of 1.8 A by X-ray diffraction. A fluorinated slow substrate forms an alpha-D-glycopyranosyl linkage to one of the two invariant carboxylates, Glu 233, as supported in solution by 19F-NMR studies. The resulting ester linkage is coplanar with the cyclic oxygen of the proximal saccharide and is inferred to form a strong hydrogen bond with the 2-hydroxyl of that saccharide unit in natural substrates. The active-site architecture of this covalent intermediate gives insights into both the classical double-displacement catalytic mechanism and the basis for the enzyme's specificity.

A new method that uses nonradioactive active site-directed enzyme inactivators and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESIMS/MS) to identify labeled peptides in a proteolytic digest is described. This method relies upon the fragmentation of labeled peptides in a predictable and reproducible manner in the collision cell of a tandem mass spectrometer. The exoglycanase from Cellulomonas fimi, endoglucanase C from Clostridium thermocellum, and the beta-glucosidase from Agrobacterium faecalis were labeled using 2-deoxy-2-halo-beta-glycosides, digested with pepsin, and subjected to HPLC-ESIMS/MS analysis, scanning in the neutral loss mode. Under these conditions only peptides that lose the (known) mass of the label are detected. Preliminary identification of candidate peptides can be achieved from the mass measured, in combination with the known sequence of the protein. Peptide identity can be confirmed through subsequent sequencing, either via further tandem MS experiments or via the Edman degradation. In all cases the peptides identified in this manner were consistent with those identified by the standard radioactive method. This mass spectrometric method represents a rapid, nonradioisotopic solution to the problem of identifying a modified peptide in a complex mixture. The technique is also sensitive, requiring only picomole amounts of protein.

The exoglucanase/xylanase from Cellulomonas fimi (Cex) has been subjected to a detailed kinetic investigation with a range of aryl beta-D-glycoside substrates. This enzyme hydrolyzes its substrates with net retention of anomeric configuration, and thus it presumably follows a double-displacement mechanism. Values of kcat are found to be invariant with pH whereas kcat/Km is dependent upon two ionizations of pKa = 4.1 and 7.7. The substrate preference of the enzyme increases in the order glucosides < cellobiosides < xylobiosides, and kinetic studies with a range of aryl glucosides and cellobiosides have allowed construction of Broensted relationships for these substrate types. A strong dependence of both kcat (beta 1g = -1) and kcat/Km (beta 1g = -1) upon leaving group ability is observed for the glucosides, indicating that formation of the intermediate is rate-limiting. For the cellobiosides a biphasic, concave downward plot is seenj for kcat, indicating a change in rate-determining step across the series. Pre-steady-state kinetic experiments allowed construction of linear Broensted plots of log k2 and log (k2/Kd) for the cellobiosides of modest (beta 1g = -0.3) slope. These results are consistent with a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbonium ion-like transition states. Secondary deuterium kinetic isotope effects and inactivation experiments provide further insight into transition-state structures and, in concert with beta 1g values, reveal that the presence of the distal sugar moiety in cellobiosides results in a less highly charged transition state.(ABSTRACT TRUNCATED AT 250 WORDS)

Several fluorinated oligosaccharides, including 2-deoxy-2-fluoro derivatives of cellobiose, maltose, and maltotriose were synthesized by the action of fluorine or acetyl hypofluorite on the corresponding glycal peracetates. Temperature effects on the stereoselectivities of these reactions were examined. Addition of acetyl hypofluorite to several 2-substituted glycals in the gluco or galacto series gave 2,2-disubstituted arabino- or lyxo-hexose derivatives; 3,4,6-tri-O-acetyl-2-fluoro-D-glucal or the analogous galactal yielded 2-deoxy-2,2-difluoro arabino- or lyxo-hexose peracetates, whereas 2-acetoxy-3,4,6-tri-O-acetyl-D-glucal or the analogous galactal gave 2(R)-2-acetoxy-2-fluoro-arabino- or lyxo-hexose peracetates, respectively. 2-Acetamido-3,4,6-tri-O-acetyl-D-glucal gave 2(R)-2-acetamido-2-acetoxy-3,4,6-tri-O-acetyl-alpha-D-arabino-hexopyrano syl fluoride. 2,4-Dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside was an inactivator of the exoglucanase from Cellulomonas fimi while 2-deoxy-2-fluoro-alpha-maltosyl and alpha-maltotriosyl fluorides were slow substrates of human pancreatic alpha-amylase and rabbit muscle glycogen debranching enzyme, respectively.

A new mechanism-based inactivator of beta-1,4-glucanases, 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-cellobioside, was synthesized and used to trap the intermediate formed during catalysis by endoglucanase C (CelC) from Clostridium thermocellum. Ion spray mass spectrometry confirmed the 1:1 stoichiometry of the incorporation of the inactivator into the enzyme. Inactivation followed the required pseudo first-order kinetic behavior and kinetic parameters for the process were determined. Although the intermediate trapped was relatively stable (t1/2 = 25 h), turnover was facilitated by transglycosylation following the addition of phenyl-beta-D-thiocellobioside and cellobiose, thus demonstrating the catalytic competence of the trapped intermediate. The nucleophilic amino acid residue involved was identified as Glu280 by labeling the enzyme with tritiated inactivator, cleaving it into peptides and sequencing the radiolabeled peptide. Ion spray mass spectrometric analysis of the peptide confirmed the sequence and the mode of attachment of the sugar to the peptide. Alignment of all known related beta-1,4-glucanases showed that Glu280 is strictly conserved in family A enzymes, consistent with its key role in catalysis.

In addition to its known substrate activity with p-nitrophenyl beta-cellobioside, the exoglucanase from Cellulomonas fimi has been shown to utilize substituted phenyl beta-glucosides as substrates, of which the best is 2',4'-dinitrophenyl beta-D-glucopyranoside. The enzyme can be inactivated by treatment with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside, by trapping of the covalent intermediate in catalysis, as has been shown for a beta-glucosidase (Withers, S.G., and Street, I.P. (1988) J. Am. Chem. Soc. 110, 8551-8553). The intermediate formed is stable but can undergo turnover in the presence of cellobiose, reactivating the enzyme by transglycosylation. Using a tritium-labeled inactivator it has been possible to isolate and sequence a radiolabeled peptide from this enzyme, and the active site nucleophile has been identified as glutamic acid residue 274. This glutamic acid residue and its sequentially proximal amino acids are absolutely conserved in the homologous family F of cellulases.