Saul Roseman

Physiological aspects of chitin catabolism in marine bacteria.

Chitin, a carbohydrate polymer composed of alternating beta-1, 4-linked N-acetylglucosamine residues is the second most abundant organic compound in nature. In the aquatic biosphere alone, it is estimated that more than 10(11) metric tons of chitin are produced annually. If this enormous quantity of insoluble carbon and nitrogen was not converted to biologically useful material, the oceans would be depleted of these elements in a matter of decades. In fact, marine sediments contain only traces of chitin, and the turnover of the polysaccharide is attributed primarily to marine bacteria, but the overall process involves many steps, most of which remain to be elucidated. Marine bacteria possess complex signal transduction systems for: (1) finding chitin, (2) adhering to chitinaceous substrata, (3) degrading the chitin to oligosaccharides, (4) transporting the oligosaccharides to the cytoplasm, and (5) catabolizing the transport products to fructose-6-P, acetate and NH(3). The proteins and enzymes are located extracellularly, in the cell envelope, the periplasmic space, the inner membrane and the cytoplasm. In addition to these levels of complexity, the various components of these systems appear to be carefully coordinated by intricate regulatory mechanisms.

The bacterial phosphoenolpyruvate: Sugar phosphotransferase system

The bacterial phosphotransferase system participates in diverse physiological phenomena; its best characterized function is in the group translocation of sugars that are substrates of the system. Such sugars are phosphorylated as they are translocated across the cell membrane. Isolation of different proteins of the phosphotransferase system and reconstitution of the complex shows that in the net transfer of the phosphoryl group from phosphoenolpyruvate to a given sugar the phosphoryl group is sequentially transferred from one protein to another. In all cases so far studied, with one important exception, the phosphoryl group is linked to the proteins through a nitrogen atom in the imidazole ring of a histidyl residue. In the exceptional protein, the phosphoryl group is linked to a carboxy group. An additional function of the phosphotransferase system is to regulate the uptake of sugars that cannot be phosphorylated.

Understanding glucose transport by the bacterial phosphoenolpyruvate:glycose phosphotransferase system on the basis of kinetic measurements in vitro.

The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected. To address the issue of whether the behavior in vivo of the PTS can be understood in terms of these enzyme kinetics, a detailed kinetic model was constructed. Each overall phosphotransfer reaction was separated into two elementary reactions, the first entailing association of the phosphoryl donor and acceptor into a complex and the second entailing dissociation of the complex into dephosphorylated donor and phosphorylated acceptor. Literature data on theK m values and association constants of PTS proteins for their substrates, as well as equilibrium and rate constants for the overall phosphotransfer reactions, were related to the rate constants of the elementary steps in a set of equations; the rate constants could be calculated by solving these equations simultaneously. No kinetic parameters were fitted. As calculated by the model, the kinetic parameter values in vitro could describe experimental results in vivo when varying each of the PTS protein concentrations individually while keeping the other protein concentrations constant. Using the same kinetic constants, but adjusting the protein concentrations in the model to those present in cell-free extracts, the model could reproduce experiments in vitro analyzing the dependence of the flux on the total PTS protein concentration. For modeling conditions in vivo it was crucial that the PTS protein concentrations be implemented at their high in vivo values. The model suggests a new interpretation of results hitherto not understood; in vivo, the major fraction of the PTS proteins may exist as complexes with other PTS proteins or boundary metabolites, whereas in vitro, the fraction of complexed proteins is much smaller.

The Chitin Catabolic Cascade in the Marine Bacterium Vibrio furnissii

We have described some steps in chitin catabolism by Vibrio furnissii, and proposed that chitin oligosaccharides are hydrolyzed in the periplasmic space to GlcNAc and (GlcNAc)2. Since (GlcNAc)2 is an important inducer in the cascade, it must resist hydrolysis in the periplasm. Known V. furnissii periplasmic hydrolases comprise an endoenzyme (Keyhani, N. O. and Roseman, S. (1996) J. Biol. Chem. 271, 33414-33424), and the β-N-acetylglucosaminidase, ExoI, reported here. ExoI was isolated from a recombinant strain of Escherichia coli, and hydrolyzes aryl-β-GlcNAc, aryl-β-GalNAc, and chitin oligosaccharides. No other β-GlcNAc glycosides were cleaved. The pH optimum was 7.0 for (GlcNAc)n, n = 3-6, but 5.8 for (GlcNAc)2. At the pH of sea water (8.0-8.3), the enzymatic activity with (GlcNAc)2 is virtually undetectable. These results explain the stability of (GlcNAc)2 in the periplasmic space. The cloned β-GlcNAcidase gene, exoI, encodes a 69,377-kDa protein (611 amino acids); the predicted N-terminal 20 amino acid residues matched those of the isolated protein. The protein amino acid sequence displays significant homologies to the α- and β-chains of human hexosaminidase despite their marked differences in substrate specificities and pH optima.

Rapid and sensitive determination of sphingosine bases and sphingolipids with fluorescamine.

Abstract A rapid and sensitive method is described for determining sphingosine and sphingolipids in the 1–100 nanomole range. Sphingosine is released from the sphingolipids by hydrolysis with hydrochloric acid in aqueous methanol, and then reacted with fluorescamine at pH 8.0. The same fluorescence intensities were obtained with equimolar concentrations of sphingosine, psychosine, cerebroside, and sphingomyelin. A hexosamine-containing sphingolipid, ganglioside, gave about twice the expected fluorescence. This result is explained by the fact that hexosamines and other primary amines react with fluorescamine. However, the method was easily modified to determine sphingosine in gangliosides by extracting the hydrophobic base from the hydrolysis mixture with ether. The procedure should have broad application in the field of sphingolipid chemistry and biochemistry.

A sodium-dependent sugar co-transport system in bacteria*

Summary A characteristics difference between active transport systems in bacterial and animal cells is that the latter usually require sodium ions and operate by the process of co-transport. The melibiose permease system (TMG permease II) of Salmonella typhimurium has now been identified as a sodium-dependent co-transport system. Co-transport, and the analogous mechanism, counter-transport, may be the underlying mechanisms for the active transport of many solutes by bacterial cells, although they may be difficult to detect.

The Chitin Catabolic Cascade in the Marine Bacterium Vibrio furnissii MOLECULAR CLONING, ISOLATION, AND CHARACTERIZATION OF A PERIPLASMIC CHITODEXTRINASE

Chitin catabolism in Vibrio furnissii comprises several signal transducing systems and many proteins. Two of these enzymes are periplasmic and convert chitin oligosaccharides to GlcNAc and (GlcNAc)2. One of these unique enzymes, a chitodextrinase, designated EndoI, is described here. The protein, isolated from a recombinant Escherichia coli clone, exhibited (via SDS-polyacrylamide gel electrophoresis) two enzymatically active, close running bands (~mass of 120 kDa) with identical N-terminal sequences. The chitodextrinase rapidly cleaved chitin oligosaccharides, (GlcNAc)4 to (GlcNAc)2, and (GlcNAc)5,6 to (GlcNAc)2 and (GlcNAc)3. EndoI was substrate inhibited in the millimolar range and was inactive with chitin, glucosamine oligosaccharides, glycoproteins, and glycopeptides containing (GlcNAc)2. The sequence of the cloned gene indicates that it encodes a 112,690-kDa protein (1046 amino acids). Both proteins lacked the predicted N-terminal 31 amino acids, corresponding to a consensus prokaryotic signal peptide. Thus, E. coli recognizes and processes this V. furnissii signal sequence. Although inactive with chitin, the predicted amino acid sequence of EndoI displayed similarities to many chitinases, with 8 amino acids completely conserved in 10 or more of the homologous proteins. There was, however, no “consensus” chitin-binding domain in EndoI.

Modified assay procedures for the phosphotransferase system in enteric bacteria.

Abstract Conditions for the assay of individual components of the bacterial phosphotransferase system (PTS) are presented wich offer two important improvements over earlier methods. First, a lactate dehydrogenase-coupled assay for phosphocarrier proteins (HPr, FPr, and Factor IIIGle) which permits their measurement in either pure or partially pure form was developed. Quantitation by this assay does not rely on the level of activity of the enzymes used. Second, conditions under which Enzyme I activity was proportional to enzyme concentration are given. With these methods levels of PTS components have been measured that are 2-to 20-fold higher than those previously reported. These levels can now account for various PTS functions measured in vivo. Further, we have shown that the phosphocarrier proteins HPr and Factor IIIGle are substrates for their respective enzymes which show typical Michaelis-Menten kineties. In addition, a method for the partial purification of Enzyme II-BGle essentially free of Enzyme IIMan activity is presented.

Chitin catabolism in the marine bacterium Vibrio furnissii. Identification, molecular cloning, and characterization of A N, N'-diacetylchitobiose phosphorylase.

The major product of bacterial chitinases isN,N′-diacetylchitobiose or (GlcNAc)2. We have previously demonstrated that (GlcNAc)2 is taken up unchanged by a specific permease inVibrio furnissii (unlike Escherichia coli). It is generally held that marine Vibrios further metabolize cytoplasmic (GlcNAc)2 by hydrolyzing it to two GlcNAcs (i.e. a “chitobiase ”). Here we report instead thatV. furnissii expresses a novel phosphorylase. The gene,chbP, was cloned into E. coli; the enzyme, ChbP, was purified to apparent homogeneity, and characterized kinetically. The DNA sequence indicates that chbP encodes an 89-kDa protein. The enzymatic reaction was characterized as follows. ( GlcNAc ) 2 + P i ⇌ GlcNAc α 1 P + GlcNAc K cq ′ = 1.0 ± 0.2 REACTION   1 TheKm values for the four substrates were in the range 0.3–1 mm.p-Nitrophenyl-(GlcNAc)2 was cleaved at 8.5% the rate of (GlcNAc)2, and p-nitrophenyl (PNP)-GlcNAc was 36% as active as GlcNAc in the reverse direction. All other compounds tested displayed ≤1% of the activity of the indicated substrates including: for phosphorolysis, higher chitin oliogsaccharides, (GlcNAc) n ,n = 3–5, cellobiose, PNP-GlcNAc, and PNP-(GlcNAc)3; for synthesis, (GlcNAc) n (n = 2–5), glucose, etc. (GlcNAc)2 is a major regulator of the chitin catabolic cascade. Conceivably GlcNAc-α-1-P plays a similar but different role in regulation.

Molecular cloning and characterization of a novel beta-N-acetyl-D-glucosaminidase from Vibrio furnissii.

The accompanying papers (Keyhani, N. O., and Roseman, S. (1996) J. Biol. Chem. 271, 33414-33424; Keyhani, N. O., and Roseman, S. (1996) J. Biol. Chem. 271, 33425-33432) describe two unique β-N-acetylglucosaminidases from Vibrio furnissii. A third, ExoII, is reported here. The gene, exoII, was cloned into Escherichia coli, sequenced, and ExoII purified to apparent homogeneity (36 kDa). The molecular weight and N-terminal 16 amino acids of the protein conform to the predicted sequence. ExoII exhibited unique substrate specificity. It rapidly cleaved p-nitrophenyl and 4-methylumbelliferyl β-GlcNAc, was slightly active with p-nitrophenyl-β-GalNAc, and was inactive with all other GlcNAc derivatives tested, including N,N′-diacetylchitobiose and (GlcNAc)n, n = 3-6. Unlike GlcNAc (Ki, 210 μM), (GlcNAc)n are poor inhibitors of ExoII. The predicted protein sequence is unique among β-N-acetylglucosaminidases excepting Cht60, recently cloned from a marine Alteromonas (Tsujibo, H., Fujimoto, K., Tanno, H., Miyamoto, K., Imada, C., Okami, Y., and Inamori, Y. (1994) Gene (Amst.) 146, 111-115). Cht60, a chitobiase, is 26.9% identical to ExoII in a 182-amino acid overlap, but the two enzymes differ in substrate specificity and other properties. ExoII shares similarity with five bacterial and yeast β-glucosidases, up to 44% identity in the 25-amino acid catalytic domain. By analogy, ExoII may play a role in signal transduction between invertebrate hosts and V. furnissii.