Biochemistry of chitin synthase.

This article compiles the papers dealing with the biochemistry of chitin synthase (CS) published during the last decade, provides up-to-date information on the state of knowledge and understanding of chitin synthesis in vitro, and points out some firmly entrenched ideas and tenets of CS biochemistry that have become of age without hardly ever having been critically re-evaluated. The subject is dealt with under the headings "Components of the CS reaction" (educt, cation requirement and intermediates; product), "Regulation of CS" (cooperativity and allostery; non-allosteric activation or priming of CS; latency), "Concerted action of CS and enzymes of chitinolysis", "Inhibition of CS", "Multiplicity of CSs", and "Structure of CS" (the putative UDPGlcNAc-binding domain of CS; identification of CS polypeptides; glycoconjugation). The prospects are outlined of obtaining a refined three-dimensional (3D) model of the catalytic site of CS for biotechnological applications.

Beta-N-acetylhexosaminidase: a target for the design of antifungal agents.

This review provides biochemical, analytical, and biological background information relating to beta-N-acetylhexosaminidase (HexNAc'ase; EC 3.2.1.52) as an emerging target for the design of low-molecular-weight antifungals. The article includes the following: (1) a biochemical description of HexNAc'ase (reaction catalyzed, nomenclature, and mechanism of action) that sets it apart from other, similar enzymes; (2) an overview and a critical evaluation of methods to assay the enzyme, including in crude extracts (photo- and fluorometric procedures with model substrates; HPLC/pulsed amperometric detection of N-acetylglucosamine and chito-oligomers; end-point vs. rate measurements); (3) a summary of some general characteristics of HexNAc'ases from fungi and organisms of other types (Km values, substrate preference, and glycoconjugation); (4) an hypothesis of a specific target function of wall-associated HexNAc'ase (a component of the assembly of surface-located enzymes effecting a continuous turnover and remodelling of the wall fabric through its combined hydrolytic and transglycosylating activities, and a mediator enzyme acting in concert with chitinase and chitin synthase to provide for the controlled lysis and synthesis of chitin during growth); (5) a tabulation of the structural formulae of reaction-based HexNAc'ase inhibitors with Ki values < or = 100 microM (some of them representing transition state mimics that could serve as leads for the development of new antifungals); and (6) an outline of approaches towards the establishment of a three-dimensional model of HexNAc'ase suitable for a truly rational design of antimycotics as well as agricultural fungicides.

Stereochemical requirements of chitin synthase for ligand binding at the allosteric site for N-acetylglucosamine.

The substrate kinetics of chitin synthase (CS) were non-Michaelian, irrespective of the type of enzyme preparation studied (105-S chitosomal CS, and 16-S ex walls), even in the presence of saturating GlcNAc. An unexplained idiosyncrasy of this enzyme, which is likely to be responsible for this phenomenon, is evident from the striking non-linearity of product deposition with time at low substrate or low enzyme concentrations, particularly in the absence of GlcNac. The possibility can be excluded that this non-linearity is due to the formation of soluble by-products or intermediates in the form of chito-oligomers, as shown by HPLC/pulsed amperometric detection analysis. Additional evidence was sought for the tenet that CS is homotropically-heterotropically regulated, at least under steady-state reaction conditions. Substrate kinetic curves established from rate data for the linear reaction phase only were used for modelling. These could be reasonably well parameterised on the basis of the Monod mathematical model for co-operative ligand binding. Within a series of test compounds used to assess the stereochemical conditions of the allosteric site of CS for effector binding, N-acetylglucosaminono-1,5-lactone oxime excelled. Requirements for effector binding are as follows: (a) an aminoglucopyranose skeleton with the amino function acetylated, and (b) a single-bonded oxo-function present at C(1), which is preferentially a hydrogen bond donor, that may be equatorially spaced off, but must not be alpha-anomeric. The implications of these findings for chitin synthesis in vivo are discussed in terms of a mechanistically based fitness of CS to operate efficiently under vastly different combinations of substrate could be coordinately linked to the catabolism of chitin.

Inhibition of beta-N-acetylglucosaminidase by glycon-related analogues of the substrate.

Inhibition studies on beta-N-acetylglucosaminidase (EC 3.2.1.30) of widely differing origins (animal, plant, fungus) were carried out with N-acetylglucosaminono-1,5-lactone (1), N-acetylglucosaminono-1,5-lactam (2), 1,5-imino-N-acetylglucosaminitol (3), and N-acetylglucosaminono-1,5-lactone oxime (4). The inhibition was competitive in all cases, and Ki values were generally in the range of 0.15-2 microM, except for the fungal enzyme (5-20 microM). To assess the kinetics of enzyme-inhibitor complex formation, continuous enzyme activity monitoring was done with 3,4-dinitrophenyl-beta-N-acetylglucosaminide as the substrate. A slow approach to the binding-equilibrium in the time scale of minutes could not be observed with any of the inhibitors tested (1-4). The results are evaluated as to the bearing of the enzyme source on best performance of the test compounds, the sub-type of inhibition mechanism is discussed, and suggestions are made for further analogue syntheses as well as potential applications of 1-4 (particularly the O-phenylcarbamoyl derivative of the latter) in biological and medical research.

A complex chitinolytic system in exponentially growing mycelium of Mucor rouxii: properties and function.

Enzymological evidence has been sought for the purported involvement of chitinolysis in vegetative growth of filamentous fungi. A procedure has been developed for the production of fast growing and morphologically homogeneous exponential phase mycelium of the non-septate dimorphic zygomycete Mucor rouxii. A partially purified extract of this material has been subjected to gel-permeation chromatography and the chitinolytic activity of eluate fractions has been assessed using colloidal and nascent chitin and 3,4-dinitrophenyl tetra-N-acetylchitotetraoside [3,4-DNP-(GlcNAc)4] as substrates. Exponentially growing (td = 1.1 h) mycelium consisting of single short-branched hyphae contains at least seven chitinases. The two particulate ones have not been studied in detail. The soluble chitinases hydrolyse (pseudo)chito-oligomers by random cleavage of internal beta-1,4-bonds (and not by processing) and have a minimum chain-length requirement of n = 4. They are clearly distinct from beta-N-acetylglucosaminidase (beta-GlcNAc'ase) with respect to their chromatographic behaviour, substrate chain-length specificity, inhibition by chitobionolactone oxime (Ki = 175 microM), and non-inhibition by the specific beta-GlcNAc'ase inhibitor N-acetylglucosaminono-1,5-lactone oxime. Their pH optima are similar (6.5-7.0), and all can hydrolyse 3,4-DNP-(GlcNAc)4 as well as nascent chitin. With respect to their charge, response to protease treatment, behaviour upon gel-permeation chromatography and ability to use colloidal chitin as a substrate, the soluble chitinases do, however, represent two distinct groups. Type A chitinases are acidic, display partial latency, show an unusual affinity to dextran gel and act weakly on colloidal chitin. Type B chitinases are basic (or neutral) and non-zymogenic, do not behave anomalously upon gel filtration and can degrade performed chitin. An hypothesis is presented for the function of the complex chitinolytic system of the fungal hypha in branching and, possibly, also in apical growth.

N-acetylglucosaminono-1,5-lactone oxime and the corresponding (phenylcarbamoyl)oxime. Novel and potent inhibitors of beta-N-acetylglucosaminidase.

Using N-acetylglucosaminono-1,5-lactone (1) as the reference, the inhibitory activity of its (formal) derivatives N-acetylglucosaminono-1,5-lactone oxime (2) and N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)-oxime (3) was tested against beta-N-acetylglucosaminidase of different origins (animal, plant, fungus). Displaying inhibition constants of 0.45 microM and 0.62 microM, for the animal and plant enzyme, respectively, the simple oxime 2 was about equally potent as the parent lactone 1, and 50-400 times more efficient than two recently described new beta-N-acetylglucosaminidase inhibitors. The (phenylcarbamoyl)oxime 3 performed even better, particularly with the fungal enzyme (Ki = 40 nM), i.e. was about 350 times more potent than the lactone. In all cases competitive inhibition was observed with 4-nitrophenyl-beta-N-acetylglucosaminide as the substrate. With Ki/Km ratios up to 3300 for 2 and 13,600 for 3, the mode of action of these novel inhibitors is probably that of transition state mimicry. Suggestions are made for their use as a tool in biological research.

Accumulation of acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans.

Yeast (Y) and hyphal (H) cells of Mucor rouxii and Candida albicans were cultivated in liquid media containing different carbon nutrient sources (glucose, fructose, ribose) and their free acyclic polyol and trehalose contents determined using capillary gas liquid chromatography (TMS- and OAc-derivatization). Irrespective of growth form and C-source, the fraction of the water-soluble neutral components of the cellular mass of the cultures - highly homogeneous with regard to the respective cell form produced - contained glycerol, ribitol and arabitol, in addition to trehalose. The polyols contributed 0.5-2% to the biomass of M. rouxii and 1.5-6% to that of C. albicans; the values for trehalose ranged from 0.2-11% in the former and 1-3.5% in the latter species. Mucor contained higher amounts of ribitol and arabitol in H cells and larger quantities of trehalose and glycerol in Y cells. In Candida, too, hyphae always exhibited higher ribitol contents, whereas arabitol attained higher levels in yeasts under almost any conditions - regardless of the type of medium (synthetic vs. complex), stage of culture (early vs. late log-phase) and strain used. Glycerol concentration was not correlated with the growth form; trehalose contents tended to be higher in Y cells. Taking into account the facts that C. albicans and certain Mucor species are agents of opportunistic infections and are invasive mainly in the filamentous form, and that the prospective hosts do not accumulate either of these carbohydrates, the possibility is considered of using trehalose- and polyol-metabolizing enzymes as targets for designing antifungal drugs.

The physiological role of malic enzyme in grape ripening.

The high specificity of malic enzyme (ME; EC 1.1.1.40) from grape berries (Vitis vinifera L.) for the naturally occurring L-enantiomer of malic acid, its very selective C4-decarboxylation, and certain allosteric properties, reported previously, favour the conjecture of a regulatory function of ME in fruit malic acid degradation. On the other hand, high ME activity was detected even during the acid-accumulating phase of berry development. Also, the in vitro reversibility of the reaction supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate, notably high CO2/HCO 3 (-) and NADPH/NADP ratios. However, a very limited incorporation of (14)C into malate and the uniform labeling pattern of the dicarboxylic acid after administration of [U-(14)C] alanine to grape berries before and after the onset of ripening, indicate that the 'reverse" reaction does not contribute essentially to grape malate synthesis. A regulatory mechanism mediating malic acid remetabolization on the basis of cosubstrate availability, comparable to the control of the hexose monophosphate shunt, is discussed.

Determination of C distribution in photosynthetic serine and phosphoglycerate from grape leaves.

Serine and phosphoglyceric acid are the classical marker intermediates of photorespiration and reductive carbon assimilation in C(3) plants. The present paper introduces a new and fast method for the determination of (14)C distribution in these compounds by selective elimination of C-3 (NaIO(4)) or C-1 (ninhydrin/ceric sulfate). Reproducibility of the procedure was found to be better than +/-1% upon degradation of [U-(14)C]serine and [U-(14)C]glycerate standards.Determination of labeling patterns in (dephosphorylated) phosphoglycerate and serine, extracted from grape leaf discs (Vitis vinifera L.) after administration of (14)CO(2) for 1 minute showed that (14)C distribution in serine changes considerably with changing assimilatory conditions. Interpretations of data basing on unusual labeling patterns of serine must therefore be accepted with caution.

Pathway of Photosynthetic Malate Formation in Vitis vinifera, a C(3) Plant.

The time course of intramolecular isotope distribution in phosphoglyceric acid and serine was determined after exposure of grape leaf discs (Vitis vinifera L.) to (14)CO(2) (1000 microliters per liter) for variable metabolic periods, and the labeling patterns were compared with the respective isotope distribution in the C(1-3) fragment of malic acid. The results clearly support the classical concept of a close precursor-product relationship between photosynthetic phosphoglycerate and malic acid. Under the assimilatory conditions used in this study, there was no indication of an immediate carbon transfer from serine to malate as has been suggested for C(3) plants (Kent et al. 1974 Plant Physiol 53: 491-495) because of a coincident labeling of these compounds in Vicia faba. According to our data, previous evidence in favor of this hypothetical pathway is based largely on an unusual (14)C distribution in serine, due to an extreme suppression of photorespiration, as well as on arbitrary comparisons between compounds of divergent kinetic characteristics and consequently different degrees of metabolic label randomization.

Effects of amphotericin B, nystatin, and other polyene antibiotics on chitin synthase.

The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations greater than or equal to 10 micrograms/ml, filipin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a Ki value of about 0.13 mM. the effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at approximately 50 micrograms/ml. Our findings suggest the existence of binding sites (sterols?) on the chitosome that are selective for certain polyenes and that play a role in the operation of chitin synthase. Because the minimal growth inhibitory concentrations of AmB or nystatin are lower than the concentrations that inhibit chitin synthase in vitro, the possibility of chitosomal chitin synthase being a primary target for the antifungal action of these polyenes seems unlikely.

Isolation and biochemical characterization of grape malic enzyme.

Malic enzyme (ME=L-malate: NADP oxidoreductase; E.C. 1.1.1.40) was extracted by Triton X-100-induced resolubilization of enzyme proteins which denaturize spontaneously upon homogenization of grape berry material. The purification procedure included fractionating with (NH4)2SO4, preparative IEF, and Sephadex G-100 chromatography. ME was identified by TLC of the radioactive product after supplementing the assay mixture with [(14)C]malate. Cofactor dependence, pH-optimum and affinities for substrates and cosubstrates were determined. Enzymic pI was found to be 5.8, the Hill coefficients range from 1 to 3. In malate decarboxylating direction at pH 7.4, grape ME displayed positive cooperativity toward the substrate, the curve approaching normal Michaelis-Menten-kinetics at pH 7.0. Substituting Mn(2+) for Mg(2+) not only increased maximal turnover rates, but also enzymic affinity for malate. These features were considered indicative of the regulatory properties of the enzyme. Their relevance for grape malate metabolism and fruit ripening is discussed.