Distribution of adenosine 5′-triphosphate (ATP)-dependent hexose kinases in microorganisms

BioSystems, 10 (1978) 265--282 265 © Elsevier/North-Holland Scientific Publishers Ltd.

DISTRIBUTION OF ADENOSINE 5 ' -TRIPHOSPHATE (ATP)-DEPENDENT HEXOSE KINASES IN MICROORGANISMS

JUAN A. DELVALLE and CARLOS ASENSIO

lnstituto de Enzimologfa del C.S.LC., Facultad de Medicina de la Universidad Aut6noma, Madrid-34, Spain

(Received October ~10th, 1977) (Revision received February 7th, 1978)

A systematic study of adenosine triphcephate (ATP)-dependent hexose kinases among microorganisms has been undertaken. Sixteen hexose kinases of five major types were partially purified from 12 microorganisms and characterized with respect to specificity for sugar and nucleotide substrates and Michaelis constants for the sugar substrates. Glucokinase activities that phosphorylate glucose and glucosamine and are inhibited by N-acetyl-glucosamine and xylose, were found to be present in the non-sulphur photosynthetic bacteria Rhodospirillum rubrum, the blue-green algae Anacystis montana, and the protists Chlorella pyrenoidosa and Chlamydomonas reinhardtii (green algae), Hypochytrium catenoides (Hypochytridiomycete) and Saprolegnia Iitoralis (Oomycete). The myxobacteria Stigma~ella aurantiaca contains a glucokinase activity with a different specificity pattern. Anacystis and Chlorella, besides their glucokinase activities, contain highly specific fructokinases, although that from Anacystis can also phosphorylate fructosamine; fructokinase from Anacystis has a molecular weight of 20 000, and exhibits a sigmoidal saturation curve for ATP when the Mg2+/ATP ratio is 2; this curve is transformed to a Michaelian one when under the same conditions an excess of Mg 2+ (5 mM) is added. Saprolegnia however, besides the glucokinase, contains a mannofructokinase activity that phosphorylates mannose (K m 0.06 mM) and fructose (1 raM). On the other hand, hexokinase, a low specificity enzyme, was detected in the protist Allomyces arbuscui~a (Chytridiomycete) and in fungi Mucor hiemalis and Phycomyces blakesleeanus (Zygomyce- tes), and Schizophyllum commune (Basidiomycete). Schizophyllum contains a glucomannokinase activity together with hexokinase activity.

The pattern of distribution of ATP-dependent hexose kinases among microorganisms seems to parallel that reported for biosynthetic pathways for lysine. The correlation with other biochemical parameters is also considered.

I. In t roduc t ion

Microbial adenosine 5 '- tr iphosphate (ATP)- dependen t hexose kinases phosphoryla te glucose, fructose or mannose in the 6 posit ion. This interesting family o f enzymes merits fur ther a t ten t ion in order to obtain a be t te r knowledge of their funct ional characteristics and physiological significance. The available informat ion is $~arce and even cont rad ic tory , particularly with regard to the physiological role of these en:v.ymes in bacteria in compari- son to those present in fungi. Thus, in certain bacteria, unlike: fungi, the level of some hexose kinases does no t account for the rate o f uti l ization o f the sugars involved (Sebastian and Asensio, 1967, 1972). On the o ther hand,

this group o f enzymes shows a wide spectrum of sugar substrate specificities depending upon the microbial source, which might be related to physiological significance. A com- prehensive knowledge o f those functional differences would also eventually shed some light on the evolut ionary history and relationships among existent microorganisms.

A survey of the informat ion available on these enzymes reveals tha t the fungi (Ascomy- cetes) possess a low sugar specificity hexokinase (Davidson, 1960; Mazon et al., 1975; Medina and Nicholas, 1957; Risby and Seed, 1969; Ruiz-Amil and Sols, 1961; Seed and Baquero, 1965; Sols et al., 1958) which phosphoryla tes glucose, fructose and mannose. Bacteria, however, contain one or

266

several hexose kinases with relatively high sugar specificity, such as glucokinase, fructokinase, mannokinase and mannofructokinase (Asensio, 1960; Benziman and Rivetz, 1972; Kamel et al., 1966; Sabater et al., 1972a, 1972b; Sapico and Anderson, 1967; Sebastian and Asensio, 1967, 1972). The available information in the literature is restricted to certain groups of bacteria (Enterobacteria, Actinomycetes and Pseudomonas) (Asensio, 1960; Benziman and Rivetz, 1972; Kamel et al., 1966; Sabater et al., 1972a, 1972b; Sapico and Anderson, 1967; Sebastian and Asensio, 1967, 1972), protists (Eugienoids, Trypano- somes, Rhizopod amoebas, cellular slime molds) (Baumann, 1969; Lucchini, 1971; Reeves et al., 1967; Risby and Seed, 1969; Seed and Baquero, 1965) and fungi (Ascomy- cetes and Deuteromycetes) (Davidson, 1960; Maitra, 1970; MazSn et al., 1975; Medina and Nicholas, 1957; Ruiz-Amil and Sols, 1961; Sols et al., 1958).

With this perspective we have studied the types of hexose kinases present in the major groups of microorganisms for which no information was available and have focused on hexose kinases that phosphorylate glucose, fructose or mannose. The type(s) and level(s) of these enzymes present in representatives of sulphur photosynthetic bacteria, Myxo- bacteria, blue-green algae, the protists green algae, Hypochytridiomycetes, Oomycetes and Chytridiomycetes, and the fungi Zygomycetes and Basidiomycetes were examined. The hexose kinases from the different microorganisms were partially purified and characterized. We studied the substrate specificity with respect to the sugar and phosphoryl donor, and the position of phosphorylation of the sugar. The results indicate that highly specific hexose kinases were preserved through non-sulphur photosynthetic bacteria, blue-green algae, and the protists green algae, Hypochytridiomycetes and Oomycetes. Other protists such as Chytridiomycetes, however, contain a hexokinase with low specific activity. The same enzyme also is present in the fungi Zygomycetes and Basidiomycetes. This

survey of enzymes together with the data available in the literature appears to indicate that the distribution of relatively highly specific hexose kinases, particularly glucokinase, on one hand, and relatively unspecific hexokinases, on the other hand, correlates very well with the two existing types of biosynthetic pathways for lysine (Vogel et al., 1970). Other biochemical parameters such as NAD-linked glutamic and D(--)-NAD- linked lactate dehydrogenases, studied particularly in protists and fungi (L~ John, 1971, 1974) appear also to correlate with the distribution of ATP~ependent hexose kinases.

2. Materials and Methods

2.1. Microorganisms

The strains Saprolegnia litoralis (2345), Phycomyces blakesleeanus (2421), Allomyces arbuscula (2681), Mucor hiemalis (2256), Pythium debarianum (2603), Schizophyllum commune (2650), and Aerobacter aerogenes (196), were obtained from the Colecci6n Espafola de Cultivos Tipo (CECT). The numbers in parentheses correspond to the nomenclature given by CECT. Stigmatella aurantiaca strain sga 1 was kindly provided by Dr. H. Reichenbach (Botanisches Institut der Universit~t, Lehrstuhl f~ir Mikrobiologie, Freiburg, Germany). Rhodospirillum rubrum was from Dr. J.M. Ramirez de Verger (Institute of Cellular Biology CSIC, Madrid, Spain). Hypochytrium catenoides strain Karling CBS 968-69 was purchased from the Centraal- bureau voor Schimmelcultures-Baam, Holland. Anacystis montana vat. "f minor" (LB 1405/3), Chlorella pyrenoidosa strain Chick "8 H" (211/8h), and Chlamydomonas reinhardtii strain 11/32 wild type + were from Dr. M. Rodriguez-Lopez (Marafdn Institute, CSIC, Madrid, Spain); the indications in parentheses correspond to the nomenclature given by the Collection of Algae and Protozoa (Botany School, University of Cambridge, England) from where they originally came.

267

The classification system used is that of Margulis (1974) based on Whittaker's five- kingdom system (Whittaker, 1969).

2.2. Growth of microorganisms

The bacteria and protists were systemati- cally grown on glucose, fructose and mannose {0.5% for the bacteria and 1% for the other microorganisms) as carbon and energy sources, except the algae and Allomyces arbuscula which were grovm in the absence of sugars. In all cases the cultures used were checked and shown to be free of contamination by phase contrast microscopy and by growth on solid media.

The microorganisms were grown in the following media: Rhodospirillum in medium S (Lascelles, 1956); Stigmatella in the medium described by Reichenbach and Dworkin, 1969; Aerobacter in the system utilized by Kamel et ah, 1966; Anacystis in medium D (Kratz and Myers, 1955); Chlorella as described by Rodriguez..Lopez, 1966; Chlamydomo- nas in medium I (Sager and Granick, 1953); Phycomyces in that of Bergman et al., 1969; Saprolegnia as Phycomyces but without vitamins and with the addition of D,L- methionine (to a :final concentration of 0.2%), hydrolyzed casein (0.2%) and yeast extract (0.4%). Mucor was grown in the medium used by Margulis and Vishniac, 1961; Allomyces in medium YpSS (Emerson, 1941); and Pythium, Hypoci~ytrium and Schizophyllum were grown in medium GAE (Garcia Mendoza and Villanueva, 1962). All microorganisms except algae were grown with shaking in 2-liter flasks containing 500 ml of media. Bacteria were cultured at 30°C, and the remaining microorganisms except algae at 25°C. Algae were cultured in 5-liter flasks containing 2--3 liters of media at room tem- perature under a L-Fluora 20W/77 lamp, in the presence of a mixture of 5% CO2--95% air.

2.3. Perparation of crude extracts

The cells were harvested during the expo-

nential phase of growth by centrifugation in a Sorvall model RC2-B centrifuge at 18 000 × g for 10 rain (the algae were harvested by continuous centrifuging), and washed with distilled water. The cells were ground with a quanti ty of alumina (Sigma type 305) corresponding to 1/3 of the wet weight of cells, in a buffer containing: 50 mM potassium phosphate pH 7.5, and I mM disodium ethyl- endiaminetetraacetic acid (EDTA) ("phosphate" buffer), or 50 mM Tris-(hydroxy- methyl) aminomethane-HC1 pH 8.0 and I mM EDTA ("Tris" buffer). In some cases the cells were broken with a disruptor RIBI model RF-1 (Sorvall) in phosphate buffer, at 20 000 p.s.i. (pounds/in 2 ).

The resulting whole homogenates, in all cases, were centrifuged at speeds sufficient to obtain a clear supematant (referred to as "crude extract").

2.4. Analytical methods

For the measurement of the hexose kinases activities two spectrophotometric methods were used: one (Method A) coupled the production of hexose-6-phosphate to the reduction of nicotinamide adenine dinucleotide phosphate (NADP); the other (Method B) coupled the production of adenosine diphos- phate (ADP) to the oxidation of reduced nicotinamide adenine dinucleotide (NADH). In Method A, the assay mixture for glucokinase contained (~mol/ml): imidazol-HC1 (pH 7.5), 50; NADP, 0.5; hexose, 1, 5 or 50; ATP, 2.5 or 5; MgC12, 5 or 10; and 0.7 units of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). When fructokinase was assayed, 3 units of glucose phosphate isomerase (EC 5.3.1.9) were added. For the mannokinase assay, 3 units of glucose phosphate isomerase and mannose phosphate isomerase (EC 5.3.1.8) were added. In Method B the assay mixture contained (/~mol/ml): imidazol-HCl (pH 7.5), 50; KC1, 100; phosphoenolpyruvate, 2; NADH, 0.4; 1.4 units of pymvate kinase (EC 2.7.1.40); 1.4 units of lactate dehydrogenase (EC 1.1.1.27), and amounts of sugar,

268

ATP and MgCI 2 as indicated in Method A. In the sugar specificity studies, the sugars were assayed as substrates by Method A (for glucose, fructose and mannose) and Method B (for the remaining sugars), and as inhibitors by Method A. The phosphoryl donor (ATP/ Mg 2÷ = 0.5) concentration was 10 times the corresponding K m for the different hexose kinases. The K m (or Ki) and Vma x of the sugars used as substrates were determined by the Lineweaver-Burk plot. Activities of 6- phosphogluconate dehydrogenase (EC 1.1.1. 43), glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, mannosephos- phate isomerase (EC 5.3.1.8), phosphogluco- mutase (EC 2.7.5.1), and a presumed pho~ phofructo (or manno) mutase were measured by Method A. Adenosine triphosphatase activity was determined by measuring the appearance of ADP from ATP, with (pmoles); ATP, 5 ; MgC12, 10; KC1, 100; ph6sphoenol- pyruvate, 2; NADH, 0.4, and 1.4 units of pyruvate kinase and lactate dehy.drogenase. Adenylate kinase (EC 2.7.4.3) was assayed by coupling the formation of ATP t o ' t h e reduction of NADP in a system that con- talned (#moles): ADP, 5; MgC12, 10; glucose, 5; NADP, 0.5; and 0.7 units of glucose-6- phosphate dehydrogenase and yeast hexokinase {EC 2.7.1.1). Glutamic~xalacetic transaminase (EC 2.6.1.1.) was assayed by following the oxidation of NADH to nicotinamide adenine dinucleotide {NAD) with (#moles): a-ketoglutarate, 1; L-aspartate, 1; NADH, 0.3; and 1.5 units of malate dehydrogenase (EC 1.1.1.37).

Changes in the concentrations of NADH and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were followed at 340 nm in a Gilford 2400, Cary model 15 or a Beckman DB spectrophotometer. All spectrophotometric assays were carried out at 25°C. One unit of activity is defined as the amount of enzyme that catalyzes the transfor- mation of 1 ~mol of substrate/minute under the assay conditions. The specific activity is expressed in units (or milliunits) per mg of protein. The method of Lowry et al., 1951 was used for the measurement of proteins.

2.5. Purification o f the hexose kinases

Purification by gel filtration was carried out on Sephadex G-200 columns, operated between 0 and 4°C in "phosphate" buffer. Hemoglobin type I was used as a marker on Sephadex G-100 for molecular weight and was measured by absorption at 414 rim. Glucose-6-phosphate dehydrogenase, adenylate kinase and glutamic~xalacetic transaminase were also used as markers and were assayed as described above. Blue dextran was used as an indicator of the volume of exclu- sion {V0). Its absorption was measured in a Klett-Summerson photocolorimeter with filter "66". Purifications by ion exchange chromatography were performed with diethylaminoethyl (DEAE)-ceUulose. "Tris" buffer was used in order to suspend the crude extract and equilibrate the column. A linear gradient of 0 to 1 M KC1 in a total volume of 1000 ml was used for elution. With both types of columns, fractions of 1--5 ml were collected, and fractions with highest activities pooled. In some instances, the pooled material was concentrated by addition of solid ammonium sulfate to a final concentration of 80% saturation. During the ammonium sulfate purification procedures, fractions between 0--30, 30--45, 45--55 and 55--65% saturation were collected. Solid salt was slowly added with constant stirring at 0°C. After each addition the mixture was allowed to stand for 20 rain. The precipitates obtained with ammonium sulfate were recovered by centrifugation. The pellets were resuspended in 1--3 ml of the "phosphate" or "Tris" buffer depending on the type of column (see above) used for further purification.

Heat treatments were done by heating aliquots of 1 ml at different temperatures for 5 min, followed by rapid cooling. They were centrifuged, and the supematants assayed for activity. The measurement of purified fractions {heat, ammonium sulfate or columns) was carried out by Method A.

2.6. Chemicals

The sugars used (all D configuration unless

otherwise specified were purchased from Pfanstiehl, Waukegan, Ill., except: tagatose, which was from Koch-Light, Buckinghamshire, England; glucosamine, from Nutritional Bio- chemicals Co., Cleveland, Ohio; L-sorbose and mannosamine, from Sigma Chemical Co., St. Louis, Mo. The nucleoside triphosphates and bovine hemoglobin type 1 were obtained from Sigma. The sugar phosphates, coenzymes NADP and NADH, and the auxiliary enzymes were purchased from Boehringer, Mannheim, Germany.

3. Results

3.1. Phosphorylating activity of crude extracts from the different microorganisms

Phosphorylating activity of crude extracts (in "phosphate" buffer) from the different microorganisms was determined as indicated in Material and Methods. Crude extracts with residual sugar were previously dialyzed against the same buffer (100 : 1; v/v). The phosphorylating activities found are indicated in Table 1. The microorganisms that were grown heterotrophically did not show significant differences in teJ.~ns of specific activity with respect to the sugar utilized for growth. The blue-green algae, protists and fungi examined, except Pythium and Chlamydomonas, possess activity on glucose, fructose and mannose. The bacteria studied and the protist Hypochy- trium, only showed phosphorylating activity on glucose. Marked differences in specific activity of hexose kinases from bacteria and algae in compm~on to fungi were found.

3.2. Identification of the hexose kinase involved

The crude extracts from the different microorganisms were partially purified for identification of the hexose kinases involved and for removing or lowering the enzymatic activities (adenosine triphosphate, adenylate kinase, hexose-phosphate isomerase, and 6- phosphogiuconate dehydrogenase) interfering with the methods of characterization of the

269

hexose kinases. In all cases, adequate controls were run in parallel to measure the marginal activity of those interfering enzymes to eventually make the pertinent corrections. The identification of the phosphorylating activities present in the microorganisms used is described below.

(i) Rhodospirillum rubrum. The phosphorylating activity on glucose present in the fructose.grown cell extract precipitates mostly between 30 and 45% saturation of ammonium sulfate and this fraction was used for its characterization.

(ii) Stigmatella aurantiaca. Cells obtained by culturing in a minimal medium with glucose, were broken in a RIBI disruptor (see Methods). The phosphorylating activity on glucose from the crude extract precipitates between 0 and 45% saturation of ammonium sulfate. The pellet was resuspended in the original volume, and this preparation heated at 55°C for 5 min. After centrifugation of the heated material, the supernatant showed a 4-fold increase in specific activity and a 50% recovery in respect to the crude extract.

(iii) Aerobacter aerogenes. A crude extract from cells grown on minimal medium with glucose was filtered throtlgh a column (2 X 40 cm) of Sephadex G-200. A preparation obtained by pooling the fractions with highest glucose phosphorylating activities was used for its characterization.

(iv) Anacystis montana. The crude extract exhibits phosphorylating activities on glucose, fructose and mannose. The activity on mannose was lost in 24 h, or by ammonium sulfate treatment. The other two activities could be resolved by ammonium sulfate fractionation. The glucose phosphorylating activity precipitates mostly between 30 and 55% saturation, and that of fructose between 55 and 65%. A low ghcokinase contamination of the latter fraction disappeared in 24 h, so that it was possible to characterize the fructokinase activity.

270

TABLE 1

Hexose phosphorylating activity of crude extracts from different microorganisms

Microorganism Growth conditions

Activity (mU/mg protein) on

Glucose Fructose Mannose

Prokaryotes Rhodospirillum rubrum

Stigmatella aurantiaca

Anacystis montana Chlorella pyrenoidosa

Eukaryotes Chlamydomonas reinhardtii Saprolegnia litoralis

Mucor hiemalis

Phycomyces blakesleeanus

Allomyces arbuscula Pythium debarianum

Hypochytrium catenoides

Schizophyllum commune

Glucose 40 < 1 < 1 Fructose 37 < 1 < 1 Mannose 34 < 1 < 1 Glucose 70 < 1 < 1 Fructose 50 < 1 < 1 Mannose 56 < 1 < 1 Autotrophic 10 15 6 Autotrophic 73 28 25

Autotrophic 56 5 < 1 Glucose 170 200 90 Fructose 180 260 100 Mannose 150 2 4 0 100 Glucose 1300 1690 1040 Fructose 1290 1670 1030 Mannose 1280 1660 1020 Glucose 480 380 220 Fructose 660 570 290 Mannose 660 570 330 Rich media YpSS 211 172 124 Glucose < 1 71 13 Fructose < 1 89 20 Mannose < 1 134 32 Rich media YpSS 2980 < 1 < 1 Glucose 2600 < 1 < 1 Fructose 2360 < 1 < 1 Mannose 3090 < 1 < 1 Glucose 170 220 120 Fructose 650 700 490 Mannose 330 210 170

The activities were measured by Method A with 50 mM hexose and 5 mM ATP. The values represent the mean of at least 3 experiments.

(v) Chlorella pyrenoidosa. The crude extract showed phosphorylating activity on glucose, fructose and mannose. As in the case of Anacystis, the activity on mannose disap- pears in 24 h or by ammonium sulfate fractionation. The phosphorylating activity on glucose was partially resolved from that on fructose by filtration of 2 ml crude extract (4.8 mg protein/ml and 131 units/mg protein

of glucose phosphorylating activity) through a column (1.5 × 30 cm) of Sephadex G-200. Fractions (1.2 ml) with highest glucose phosphorylating activity and free of f~uctokinase contamination were pooled for characterization of this activity. The f~uctose phosphorylating activity was totally separated from that on glucose, and eventually characterized by filtration through Sephadex G-100 (Fig. la).

271

4o

E 3O

E

C]. ;/ucok,nose ~

2 i Z tO 20 30 40

FRACTION NUMBER

-~15 E

I0

- - 5

Monnofructokmase b. \

20 40 60 80 IO0 FRACTION NUMBER

1.00

0.75

0.50

025

8O

E

: ~ 6 0 E

~ 4 0 >

2O

c. / Port/'cu/ote g /ucok inose

20 40 60

Soluble cokinose

Monnofructok/nose

i 80

FRACTION NUMBER

2O

E

d.

c o ~ Hexokinese

H O . 5 0 ~ ' ~ /

G/uco- k~nose 0.4 M --~ ~ mann°kinoseO32M~o 25

J; ~ ' '¢" ' I I I / I0 20 50 60 70 80 90 I00 200 216

FRACTION NUMBER

Fig. 1. Separation oil hexose kinases from ChloreUa pyrenoidosa, Saprolegnia litoralis and Schizophyllum commune. In Fig. l a , the separation of glucokinase and fructokinase from Chlorellapyrenoidosa is shown: 1.2 ml of crude extract (5.5 mg prote in/ml , and 131 and 22 mU/mg protein of phosphorylat ing activity with glucose and fructose, respectively) was applied to a column (1.83 x 44 cm) of Sephadex G-100 (flow rate = 0.3 ml/min); 2.5 ml fractions were collected. Fig. l b illustrates the separation o f soluble glucokinase and mannofructokinase from Saprolegnia litoralis: 5 m] of crude extract (2.1 mg prote in /ml and phosphorylat ing activities with glucose, fructose and mannose of 43, 127 and 44 mU/mg protein, respect ively)obta ined from a homogenate of mycelia grown in glucose medium by centrifuging at 105 000 X g for 2 h , w a s chromatographed through a column (2.5 X 5.0 cm) o f DEAE-cellulose (flow rate = 1.1 ml/min) ; 5 ml fractions were collected. Samples from various regions of the elution profile of mannofructokinase gave the same relative activity with fructose and mannose as that of the crude extract . Fig. l c shows the separation o f particulate glucokinase with respect to the soluble form and of mannofructokinase from a crude extract of Saprolegnia litoralis: 4 ml of crude extract (3.5 mg prote in/ml and 220 mU of phosphozylat ing activity with glucose) obtained by centrifuging (at 28 000 x g for 60 rain) a homogenate of mycelia grown in glucose medium, was chromatographed through a column (2.5 x 50 cm) o f Sephadex G-200 (flow rate = 6.0 ml/min) ; 2 ml fractions were collected. Particulate glucokinase appears in the void volume (measured with blue dextran), followed by soluble glucokinase together with mannofructokinase. Fig. l d represents the separar.ion of glucomannokinase and hexokinase from Schizophyllum commune: 16.5 ml of crude extract (0.5 mg prote in /ml and phosphorylat ing activity with glucose, fructose and mannose o f 320, 110 and 90 mU/mg protein, respectively), from a culture grown on mannose, was precipitated (between 45 and 70%) with ammonium sulfate. ~Ihe pellet was resnspended in 2.5 ml of "Tris" buffer; this last preparat ion (0.9 mg prote in/ ml and phosphorylat ing activity with glucose, fructose and mannose of 800, 490 and 480 mU/mg protein, respectively) was chromatographed through a column (2.5 X 50 cm) o f DEAE-cellulose (flow rate = 1 ml/min). Fractions of 4.6 ml were collected. Symbols represent activities with: =, 5 mM glucose; o, 5 mM fructose; ", 1 mM mannose.

272

(vi) Chlamydomonas reinhardtii. The crude extract showed phosphorylating activity on glucose and fructose but not on mannose. Activity on fructose was lost in 24 h or by filtration through a column of Sephadex G-200. The activity on glucose was partially purified by filtration through a column (2.5 X 45 cm) of Sephadex G-200.

(vii) Saprolegnia litoralis. The phosphorylating activities on glucose, fructose and mannose, obtained in glucose-grown mycelial extracts, were resolved by DEAE-ceUulose chromatography (Fig. lb). There are two activities: one that phosphorylates glucose and another mannose and fructose. The glucokinase activity occurs in soluble and particulate forms which are separable by centrifugation at 105 000 × g for 2 h, or by filtration through a column of Sephadex G-200 (Fig. lc).

(viii) Schizophyllum commune. The pbos- phorylating activities on glucose, fructose and mannose, obtained in glucose-grown mycelial extracts, precipitated mostly between 45 and 70% saturation of ammonium sulfate. Further chromatography by DEAE-cellulose (Fig. ld) resolved two phosphorylating activities: glucomannokinase and hexokinase. A glucomannokinase/hexokinase ratio of about 2 was found in the crude extracts. The levels of both activities were calculated taking into account that glucomannokinase has no activity on fructose and that the ratio of the hexokinase activity on 50 mM fructose over that on 5 or 50 mM glucose in purified fractions is 1.8. Thus, hexokinase activity (on glucose) ffi activity on 50 mM fructose/1.8, and glucomannokinase activity (on glucose)= total activity on glucose -- hexokinase activity on glucose.

(ix) Hypochytrium catenoides, Phycomyces blakesleeanus, AUomyces arbuscula, Mucor hiemalis and Pythium debaryanum. The glucose-grown mycelial crude extracts from the

first three microorganisms were chromatographed by DEAE-cellulose and that corresponding to Mucor by Sephadex G-200. The glucose phosphorylating activity that only appears to be present in ttypochytrium is soluble and thus differs in respect to that present in Saprolegnia: crude extracts obtained by centrifugation at 800 X g for 5 rain or 105 000 X g for 120 rain, yield the same phosphorylating activity on glucose. The glucose, fructose and mannose phosphoryla- ring activities found in Phycomyces, Allomy. ces, and Mucor appeared together in the chromatographic profiles and the relative activities on these sugars remained constant in randomly selected fractions; additional kinetic data (Km and K i were the same for those sugars that were substrates) indicates that only one enzyme (hexokinase) was responsible for the three activities in each microorganism. Pythium showed activity on mannose and fructose only; a crude extract obtained by centrifugation at 800 X g for 5 rain did not show detectable phosphorylating activity on glucose. The apparent mannofructokinase present in this microorganism was not further characterized. Two facts suggest that such an enzyme is involved: (a) The ratio found between the phosphorylating activities on fructose and mannose was the same in crude extracts from mycelia grown on either glucose, fructose or mannose (see Table 2); (b) This ratio was preserved after dialysis of the crude extract for 12 h at 4°C which caused a loss of 25% of the activities on both sugars.

3.3. Sugar specificity and kinetic properties of the hexose kinases found

The sugars used as substrates or inhibitors are indicated below for each kind of hexose kinase. For all the hexose kinases studied, the inhibitions obtained were competitive with respect to the main substrate and the apparent K i values for the sugars that were also substrates were similar to the corresponding K m values.

TA

BL

E 2

Su

gar

sp

ecif

icit

y o

f th

e g

luco

kin

ases

Su

gar

R

hodo

spir

iUum

St

igm

ateU

a A

nacy

stis

C

hlor

ella

C

hlam

ydo-

Sa

prol

egni

a H

ypo

chy-

ru

brum

au

rant

iaca

m

onta

na

pyre

noid

osa

mon

as

lito

rali

s tr

ium

re

inha

rdtt

i ca

teno

ides

K m

V

ma

x K

m

Vm

ax

Km

V

ma

x K

m

Vm

ax

Km

V

max

K

m

Vm

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K m

V

max

(o

r K

i)

(or

gi)

(o

r g

i)

(or

gi)

(o

r g

i)

(or

Ki)

(o

r g

i)

Glu

cose

0

.15

1

00

0

.10

1

00

0

.10

1

00

0

.10

1

00

0

.10

1

00

0

.15

1

00

0

.15

1

00

G

luco

sam

ine

5 6

0

0.5

20

2 15

3

10

0.

7 15

0

.5

75

0

.5

65

2

-Deo

xy

-glu

cose

--

--

15

3

5

..

..

..

..

..

N

-Ace

tyl-

glu

cosa

min

e 7

0

--

0.4

--

3 --

1

--

* *

0.3

--

0

.4

--

Xy

lose

3

--

2 --

7

--

7 --

8

--

30

--

5

0

--

Fru

cto

se o

r m

ann

ose

.

..

..

..

..

..

..

.

Th

e d

ash

es i

n th

e co

lum

n

of

Vm

a x

(%)

ind

icat

e th

at n

o m

easu

rab

le a

ctiv

ity

was

det

ecte

d w

ith

50

mM

of

the

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274

(i) Glucokinuses. Glucose, fructose, mannose, glucosamine, N-acetyl-glucosamine and xylose, were assayed as substrates or inhibitors of the glucokinases found. The results are indicated in Table 2. Glucose and glucosamine were the only substrates with the exception of 2deoxyglucose which was also substrate for the glucokinase from Stz&nateZZa. Xylose and N-acetyl-glucosamine, but not tictose and mannose, were inhibitors of the glucokinases studied. The kinetic parameters for the glucokinases from Anacystis and Chlorellu, on one hand, and Suprolegniu and Hypochytrium, on the other hand, are very similar. The soluble and particulate glucokinases from SuproZegniu exhibited the same kinetic and specificity characteristics. In the assay conditions mentioned in Table 2, N-acetylglucosamine was inhibitor, but not substrate, of the glucokina.ses from Aerobucter (Ki = 15 mM) and Streptomyces violuceoruber (Ki = 2 mM) (DelValle and Sabater, unpublished data.

(ii) Fructokinuses. Eighteen different sugars were assayed as substrates or inhibitors (25 mM concentration): fructose, glucose, mannose, 2deoxy-glucose, glucosamine, xylose, N-acetyl-glucosamine, galactose, sedoheptu- lose, tagatose, mannitol, arabinose, saccha- rose, L-sorbose, lyxose, fucose, mannoheptu- lose and fructosamine. Fructose (K, = 0.3 mM) was the only substrate for the fructokinase from the green algae Chlorellu, and fructose (K, = 0.7 mM) and fructosamine

(&ll = 10 mM and 95% of V,,, in respect to fructose) were the only ones for the fructokinase from the blue-green algae Anucystis. L-sorbose (Ki = 10 mM), in the case of Anucystis, and glucosamine (Ki = 25 mM), for Chlorellu, were the only inhibitors found when assayed with fructose at a concentration around K, (0.5 mM for Anacystis and 0.25 mM for ChZorelZu). The rest of the sugars examined had a K, (or Ki) > 0.1 M and V max < 1% in respect to fructose.

(iii) Munnofructokinuse. The following

sugars were assayed as substrates or inhibitors for the mannofructokinases from Suprolegnia: fi-uctose, mannose, glucose, glucosamine, 2deoxy-glucose, N-acetylglucosamine, xylose, mannosamine, L-sorbose and lyxose. Marmose

(%I = 0.06 mM; V,,, = 30% in respect to fructose), fructose (K, = 1 mM) and glucosamine (K, = 0.25 mM; V,., = 10%) were the only significant substrates. Glucose was a very poor substrate (K, = 80 mM and V max = 5%). Mannosamine (Ki = 3mM) ~YXOS~ (Ki = 20 mM) and L-sorbose (Kl = 40 mM) were inhibit.ors.

(iv) Hexokinuses and glucomunnokinases. Glucose, fructose, mannose, 2.deoxy-glucose, glucosamine, N-acetyl-glucosamine and xylose were assayed as substrates or inhibitors of hexokinases and glucomannokinases. The results and assay conditions used for the characterization of the enzymes present in Mucor, Phycomyces, Allomyces and Schizo- phyhum are shown in Table 3. As other typical hexokinases reported in the literature, they phosphorylated glucose, fructose, mannose, 2deoxy-glucose and glucosamine. Xylose and N-acetyl-glucosamine were inhibitors. Fructose was the sugar phosphorylated most readily except in the case of the hexokinase from Phycomyces. 2.Deoxy- glucose was as effective substrate as glucose although with approx. 5 times lower affinity. Mannose and glucosamine were, in this order, less efficient substrates. N-acetyl-glucosamine, unlike xylose, was a relatively good inhibitor of the hexokinases tested, especially in the case of Allomyces (Km = 0.1 mM). The glucomannokinase isolated from Schizophyl- lum exhibited a substrate specificity similar to the hexokinases described above except that fructose was neither a substrate nor an inhibitor (Km > 0.2 M and V,,, < 1% in respect to glucose).

3.4. Phosphoryl donor specificity and kinetic properties.

ATP, GTP, ITP, CTP and UTP were assayed in all cases as phosphoryl donors. Table 4

TABLE 3

Sugar specificity of the hexokinase and glucomannokinases

275

Sugar Hexokinases Glucomanno- kinase

Mucor Phycomyces Al lomyces Schizophyilum Schizophyllum hiemalis blakesleeanus arbuscula commune commune

Km Vma x Km Vmax Km Vmax K m Vmax Km Vmax (or Ki) (or Ki) (or Ki) (or Ki) (or Ki)

Glucose 0.08 100 0.09 100 0.06 100 0.3 100 0.08 100 Fructose 0.9 230 25 85 15 100 2.5 175 - - - - Mannose 0.04 90 0.1 40 0.1 45 0.2 80 0 .4 . 100 2-Deoxy-glucose 3 I00 0.4 75 0.2 I00 1.5 95 0.15 80 Glucosamine 0.5 35 0.8 35 0.8 30 0.9 50 0.4 20 N-Acetyl-glucosamine 2 - - 2 -- 0.1 - - 2 - - 0.5 - - Xylose 15 -- 100 -- 80 -- 65 ~ 75 --

For details see footnote Table 1.

TABLE 4

K m for ATP-Mg 2÷ and relative activities for nucleoside triphosphates of ATP-dependent hexose kinases

Hexose Microorganism K m Relative activities kinase a A.TP-Mg ~÷

(raM) ATP GTP ITP CTP UTP

GK Rhodospirillum rubrum 0.1 Stigmatetla aurantiaca 0.3 Anacystis montana 0.9 b Chlorella pyrenoidosa 0.8 Chlamydomonas reinhardtii 1.0 Saprolegnia litoralis 0.6 Hypochytr ium catenoides 1.5

100 45 55 20 25 100 10 30 40 65 100 25 30 15 25 100 25 30 10 35

C C C C C

100 <5 <5 <5 <5 100 <5 5 <5 <5

FK Anacystis montana 0.4 100 50 55 85 65 Chlorella pyrenoidosa 0.7 100 15 85 35 35

Saprolegnia litoralis 1.0 100 10 5 5 10

Mucor hiemalis 0.6 Phycomyces blakesleeanus 0.2 Allomyces arbuscula 0.5 Schizophyllum commune 0.1

MFK

HK 100 35 45 45 40 100 <5 10 <5 <5 100 <5 15 <5 <5 100 10 35 10 10

GMK Schizophyllum commune 0.4 100 5 10 5 5

K m for ATP-Mg 2÷ was determined by Method A with saturating concentrations of sugar, i.e,, Mg2*/ATP ffi 2. The relative activities (% in respect to ATP) were measured by Method A with 5 mM nucleoside triphosphate and 10 mM Mg 2÷, and saturating amount of sugar. a GK, glucokinase; FK, fructokinase; MFK, mannofructokinase; HK, hexokinase; GMK, glucomannokinase. b Km was determined in the presence of Mg2÷/ATP ffi 2, plus addition o f 5 mM MgCI~. c Not determined.

276

inc ludes t he K m fo r ATP-Mg 2+ and t h e relat ive spec i f ic i ty f o r t he d i f f e r e n t nuc leos ide t r i phospha t e s .

All s a tu r a t i on curves fo r ATP-Mg 2+ were Michael ian e x c e p t in t he case o f t he f r u c t o - k inase f r o m Anacystis t h a t was s igmoid a t the ra t io Mg2+/ATP = 2 (Fig. 2). This curve is t r a n s f o r m e d to a Michael ian o n e b y add i t i on o f an excess o f Mg 2+ (5 raM). T h e f ruc t o - k inase f r o m Anacystis has a m o l e c u l a r we i gh t o f a p p r o x . 2 0 0 0 0 , w h e n d e t e r m i n e d b y f i l t r a t ion t h r o u g h S e p h a d e x G-100 , e i the r in t h e absence o r p r e sence o f add i t iona l 5 m M MgCl~ in t h e c o l u m n (Fig. 3).

A m o n g the nuc leos ide t r i p h o s p h a t e s assayed, A T P was the bes t p h o s p h o r y l d o n o r , in all cases. I T P fo l l owed in e f f i c i e n c y ( f r o m 55 t o 5%) in t h e g lucokinases . GTP, UTP and CTP were less ef fec t ive . I t is in te res t ing to n o t e t he r e m a r k a b l e s imi lar i ty b e t w e e n the p a t t e r n s o f Anacystis and Chlorella. Stigma- tella presen t s this o rde r o f e f f i c i ency : ATP, UTP, CTP, ITP, GTP, t hus d i f fe r ing in r e s pec t t o t he o t h e r g lucokinases fo r b o t h sugar and p h o s p h o r y l d o n o r spec i f ic i ty . T h e f r u c t o k i - nases f r o m Anacystis and Chlorella p re s en t a d i f f e r e n t p a t t e r n . M a n n o f r u c t o k i n a s e f r o m Saprolegnia also s h o w e d a d i f f e r e n t s p e c t r u m

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Fig. 3. Estimation of the molecular weight of fructokinase from Anacystis montana. In Fig. 3a, 3 ml of a preparation in "phosphate" buffer containing 82 mU of fructokinase activity (from a 55--65% ammonium sulfate subfraction), 1.4 U of glucose-6-phosphate dehydrogenase (G-6-PDH; mol. wt. = 128 000), 1.8 U of adenylate kinase (mol. wt. ffi 21 000) and 10 mg of hemoglobin (tool. wt. ffi 34 000) (Morris, 1964)were applied to a column (1.83 x 44 cm) of Sephadex G-100 equilibrated with the same buffer. The levels of activity of the marker enzymes were at least 10 times higher with respect to those detected in t h e ammonium sulfate subfraction, and were measured as indicated in Methods. Th~ void volume (V 0 ) was measured with 0.5% blue dextran; V e = elution volume of the indicated proteins. Fig. 3b refers to the same experiment except that Sephadex G-100 was equilibrated with "phosphate" buffer plus 5 mM MgCI 2. In this experiment 1.8 U of glutamic oxalacetic transaminase as additional marker was added (GOTA; mol. wt. ffi 90 000). Symbols represent: e, marker proteins; X, fructokinase activity.

in respect to both glucokinases and fructokinases. Hexokinases and the glucomannokinase from Schizophyllum followed the same pattern mentioned for the glucokinases, with the exception of hexokinase from Mucor which besides ATP, utilized GTP, ITP, CTP and UTP with approximately the same efficiency (35--45%).

3.5. Reaction products. The following characteristics suggest that

the hexose kinases studied phosphorylate their sugar substrates at the 6~arbon position. There is an immediate coupling of the reaction product to the assay method, which is dependent on glucose-6-phosphate dehydrogenase (for glucokinases, glucomannokinases and hexokinases), or glucose-phosphate isomerase (for fructokinases and mannofructokinases) and no hexose-l-phosphate mutase(s) activities have been detected in the enzymatic preparations.

4. Discussion

The hexose kinases found and characterized in the microorganisms used in this investiga- t ion are similar within each class to other types previously described, with the exception of the glucokinase present in the myxo- bacterium St~gmatella. This enzyme, unlike the typical bacte]Sal glucokinase, phosphorylates 2<leoxy-glucose and also exhibits an unusual specificity pattern for the phosphoryl donor.

The glucokina~es from Rhodospirillum, Anaeystis, ChloreUa, Chlarnydomonas, Sapro- legnia and Hypochytrium are very similar to those reported in the bacteria Aerobacter aerogenes (Kamel et al., 1966), Streptornyces violaceoruber (Sabater et al., 1972a), and Escherichia coll. (Asensio, 1960), and to that reported in the cellular slime mold Dictyoste- lium discoideum (Baumann, 1969}, They phosphorylate glucose and glucosamine, xylose and N-acetylglucosamine being inhibitors. ITP was the next most effective nucleoside triphosphate besides ATP. All the K m's for ATP are in the same order of magnitude. It is interesting to note the great simi- larities between the glucokinases from Anacy- stis (blue-green algae) and Chlorella {green algae) on one hand, and those from Saproleg- nia (Oomycete) and Hypochytrium (Hypo- chytridiomycete) on the other hand. The glucokinase from Saprolegnia is partially particulate, unlike the other glucokinases

277

ment ioned which are soluble. Both forms of the enzyme showed identical kinetic and specificity features. A glucokinase was reported in Acetobacter xylinum that phosphorylates mannose, although poorly, besides glucose and glucosamine and is inactive on 2<leoxy-glucose (Benziman, 1972). An enzymatic activity that phosphorylates glucose, fructose and mannose has been partially characterized from crude extracts of Euglena gracilis vat. bacillaris (Belsky and Schultz, 1962). On the other hand, the presence of a non-typical bacterial glucokinase with low affinity for glucose (Km = 5 mM) and which is activated by ortophos- phate, has been described from E. gracilis strain Z (Belsky and Schultz, 1962).

The fructokinases from the blue-green algae Anacystis and the green algae Chlorella are very similar to that present in the actinomy- cete Streptomyces violaceoruber (Sabater et al., 1972b) in respect to the sugar specificity. Fructokinases from these algae are apparently very specific for fructose, although the Anacystis type also phosphorylates fructosamine with low affinity (Km = 10 raM; Vma x = 95%). The weak inhibitions due to L-sorbose (Anacystis) or glucosamine (Chiorella) had not been detected in the fructokinase from Streptomyces, which is an enzyme of very high structural requirements. The fructokinases from the blue-green algae and Strepto- myces have also in common the positive cooperativity for ATP-Mg 2+ and the activation by excess Mg 2+.

The mannofructokinase from the Oomycete Saprolegnia is the first enzyme of this type reported in eukaryotic microorganisms, and is very similar to a hexose kinase present in Escherichia coli (Sebastian and Asensio, 1967, 1972) and Leuconostoc mesenteroides (Sapico and Anderson, 1967). The mannofructokinase that appears to be present in Pythium debaryanurn (Oomycete) was not studied in detail but seems of a similar type (see Results).

The hexokinases present in Mucor (Zygo- mycete) and in SchizophyUum (Basidiomy- cete) are rather similar to the hexokinase

278

isozyme P-I from the Ascomycete 8accharo- myces cerevisiae (Colowick, 1973; Womack et al., 1973) in their velocities for glucose, fructose and mannose. The hexokinases present in Allomyces (Chytridiomycetes) and in Phycomyces (Zygomycete), however, appear to be more similar to the hexokinase isozyme P-II from Saccharomyces. All these enzymes phosphorylate 2<leoxy~lucose and glucosamine, and are inhibited by the non- substrates xylose and N-acetyl-glucosamine. As in the case of glucokinases, ITP was the next most effective nucleoside triphosphate relative to ATP. The rest of the phosphoryl donors (CTP, UTP, GTP) were utilized to the extent of about 10% or less (with the exception of hexokinase from Mucor). In each case the K m for ATP was within the same order of magnitude (between 0.1 and 0.6 mM). The hexokinases present in the fungi Ascomycetes Neurospora crassa (Medina and Nicholas, 1957) and AspergiUus orizae (Ruiz-Amil and Sols, 1961), in the Deutero- mycete Rhodotorula glutinis (Mazon et al., 1975), and in various species of the protists flagellates Trypanosoma (Risby and Seed, 1969; Seed and Baquero, 1965), although not as well characterized as that of Saccharo- myces, can be included in the group of hexokinases. Hexokinase from Aspergillus parasiticus (Davidson, 1960) can not be included within the same group as it phosphorylates galactose (86% relative to glucose).

Glucomannokinase from the Basidiomycete Schizophyllum is very closely related to an enzyme present in Saccharomyces cerevisiae (Maitra, 1970) and Rhodotorula glutinis (called glucokinase by the authors) (MazSn et al., 1975). These microorganisms possess glucomannokinase together with hexokinase. The physiological significance of this coexis- tence is an open question, particularly because of their similarity in specificity (differing only in the specificity for fructose). In a species of Rhizopod, Entamoeba histolytica, the presence of two glucokinase isozymes with indistinguishable kinetic and specificity characteristics has been described

(Reeves et al., 1967). These glucokinases (in fact, they could be named glucomannokinases because of their specificity), are not similar to the other glucomannokinases mentioned above since they phosphorylate N-acetyl- glucosamine with good efficiency (Km = 0.05 mM and V max = 40% in respect to glucose).

The contribution of the hexose kinases here reported to the utilization of exogenous sugars used for growth is a question that remains open. Studies on the rate of utiliza- t ion of glucose, fructose and mannose by the microorganisms used, together with the specific activities found (see Table 1 and Introduction), will give insight on the physiological role of the hexose kinases detected. The observation that Rhodospirillum, Stigma. teUa, Pythium and Hypochytriurn grew in the presence of certain sugars for which there were apparently no phosphorylating activities, suggest the existence of some other mecha- nism than the ATP-hexose phosphotransferase system for the utilization of those sugars.

The current data on the different types of ATP-dependent hexose kinases present in the biological groups investigated, together with the information of this family of enzymes reported in the literature, give rise for the first t ime to a general but necessarily prelimi- nary view of the distribution of these enzymes among microorganisms (see Table 5). It is apparent that the relatively highly specific hexose kinases present in bacteria have been preserved through blue-green algae, green algae and the protists Hypochytr idomy- cetes, Oomycetes and cellular slime molds. Other protists such as Chytridiomycetes and Trypanosomes, however, exhibit the low specific enzyme hexokinase. All the fungi examined, that is, Zygomycetes, Ascomy- cetes, Basidiomycetes and Deuteromycetes, also contain hexokinase activity.

The functional approach of our study does not allow us to go further than suggesting some evolutionary relationships among the microorganisms examined.

The distribution of hexose kinases among microorganisms exhibits a close parallelism

279

TABLE 5

List of microorganisms in which ATP-dependent hexose kinases are relatively well characterized

Higher taxon Species Type of hexose kinase

Prokaryotes Monera

Purple non-sulphur bacteria Pseudomonads Myxobacteria Actinomycetes Enterobacteria

Lactic acid bacteria Blue-green algae

Eukaryotes Protists

Green algae

Euglenids Rhizopod amoebas Cellular slime molds Hypochytridiomycetes Oomycetes

Trypanosomes

Chytridiomycetes

Fungi Zygomycetes

Ascomycetes

Basidiomycetes Deuteromycetes

Athiorhodaceae Rhodospirillum rubrum * GK Pseudomonadaceae Acetobacter xyl inum a "GK", FK Polyangiaceae Stigmatella aurantiaca * "GK" Streptomycetaceae Streptomyces violaceoruber b GK, FK, MK Enterobacteriaceae Aerobacter aerogenes c GK Enterobacteriaceae Escherichia coli d GK, MFK Lactobacillaceae Leuconostoc mesenteroides e MFK Chlorococcales Anacystis montana* GK, FK

Chlorococcales Volvocales Euglenida Amoebida Dictyosteliales Hypochytriales Saprolegniales Peronosporales Kinetoplastida Kinetoplastida Kinetoplastida Blastocladiales

Mucorales Mucorales Endomycetales Sphaeriales Plectascales Plectascales Agaricales Moniliales

Chlorella pyrenoidosa* GK, FK Chlamydomonas reinhardtii* GK Euglena gracilis f "GK" Entamoeba histolytica g "GMK" Dictyostelium discoideum h GK Hypochytrium catenoides* GK Saprolegnia litoralis* GK, MFK Pythium debaryanum * MFK Trypanosoma rhodesiense i HK Trypanosoma gain biense i HK Trypanosoma equiperdum j HK Allomyces arbuscula* HK

Mucor hiemalis* HK Phycomyces blakesleeanus* HK Saccharomyces cerevisiae k HK, GMK Neurospora crassa I HK Aspergillus orizae m HK AspergiUus parasiticu sn "HK" Schizophyllum commune* HK, GMK Rhodotorula glutinis ° HK, GMK

* Species that were studied by us. a Benziman and Riw~tz, 1972. b Sabater et al., 1972a, 1972b. c Kamel et al., 1966. d Asensio, 1960; Sebastian and Asensio, 1967, 1972. e Sapico and A n d e r ~ n , 1967.

Lucchini, 1971. g Reeves et al., 1967.

h Baumann, 1969. i Seed and Baquero, 1965. J Risby and Seed, 1969. k Sols et al., 1958;Maitra, 1970. 1 Medina and Nicholas, 1957. m Ruiz-Amil and Sols, 1961. n Davidson, 1960. o Maz6n et al., 1975.

Abreviations as in Table 4. ATP-dependent hexose kinases that merely phosphorylate other sugars than glucose, fructose or mannose, are not shown. Enzymes with quotation marks refer to hexose kinases with different specificity and/or kinetic properties in respect to those frequently found within each type of hexose kinase.

280

with that of biosynthetic lysine pathways (Vogel et al., 1970). Thus, relatively highly specific hexose kinases, particularly glucokinase, and a-e<iiamino-pimelic acid (DAP) lysine pathway are found in bacteria, blue- green algae, green algae, Hypochytridiomy- ceres and Oomycetes. Cellular slime molds contain glucokinase but it is not known which type of lysine pathway they have. On the other hand, hexokinase and the a-amino adipic acid (AAA) lysine pathway are present in the Chytridiomycetes, Zygomycetes, Ascomycetes and Basidiomycetes. Trypano- somes and Rhizopod amoebas do not contain

lysine pathways. The interesting group of Euglenoids contain a non-typical glucokinase and AAA lysine pathway.

The striking divergence between Hypochy- tridiomycetes, Onmycetes and cellular slime molds on the one hand, and Chytridiomycetes, Zygomycetes, Ascomycetes and Basidiomy- ceres on the other, in respect to lysine pathways is now strengthened by the study of their hexose kinases. Differences have also been found by. using other biochemical parameters, namely control of NAD-linked glutamic dehydrogenase and D(-) lactate dehydrogenase and their sensitivity to repres-

TABLE 6

Correlation between types of ATP-dependent hexose kinases and other biochemical criteria among some protists and fungi

Type of Lysine Type of Type of Sensitivity Hydroxy- hexose path b NAD-GDH c D(-)-LDH c to glucose d proline kinase a "in wall e

Fungi Basidiomycetes Agaricales HK, GMK AAA I g -- ? ?

Ustilaginales ? AAA I g -- ? -- Ascomycetes Endomycetales HK, GMK AAA I g III No --

Pleetascales . HK AAA I ? ? -- Sphaeriales HK AAA I ? No --

Zygomycetes Mucorales HK AAA I, II I No -- Chytridio-

mycetes Blastocladiales HK AAA II I No ? Chytridiales ? AAA I I No ?

Protists Hypochytri-

diomycetes Oomycetes

Hypochytriales GK DAP IH II Yes ? Saprolegniales GK, MFK DAP IH II Yes + Leptomitales ? DAP III II Yes ? Peronosporales MFK DAP III II Yes +

Acrasiomycetes (cellular slime molds) Dictyostellales GK ? III f "II f ? ?

+, Present; --, Absent; ?, No information available. a Abbreviations as in Table 4. b AAA, a-amino adipic acid lysine pathway; DAP, a-e-diamine pimelic acid lysine pathway. From Vogel et al.,

1970. c From I.h John (1974); see this reference for description of the types of enzymes. d Data from Ih John, 1971. e Data from Crook and Johnston 1962. f These systems are not well resolved as yet.

sion of synthesis by glucose (L~ John, 1971, 1974). Table 6 includes all these biological characteristics together with that of the presence or absence of the amino acid hydroxyproline in the cell walls (Crook and Johnston, 1962). It can be seen that Hypo- chytridiomycetes and Oomycetes have the DAP lysine pathway, type III glutamic dehydrogenase, type II lactic dehydrogenase, sensitivity to glucose effect and relatively highly specific hexose kinases. Cellular slime molds exhibit similar features. On the other hand, the protists Chytridiomycetes, lower fungi Zygomycetes, and the higher fungi Ascomycetes and. Basidiomycetes, contain the AAA lysine pathway, type I or II glutamic dehydrogenases, types I or III lactate dehydrogenase (in Basidiomycetes it is apparently absent), no sensitivity to glucose, and hexokinase. The presence or absence of hydroxyproline in the cell wall has been studied in only some of the orders mentioned. Never- theless, the awtilable data reinforce the divergence.

Other biochemical parameters extensively studied in protists and fungi are cellulose, hemicellulose or chitin in the wall (Bartnicki- Garcia, 1968; Rogers and Perkins, 1968). These structural components, however, are not reliable indicators of phylogenetic relationships between very different taxa, but are useful in del~.~eating species at the generic level (Lb John, 1974).

Acknowledgements

The advice of Drs. Alberto Sols, Lynn Margulis, E.C.C. Lin and Jesfis Sebastian is greatly appreciated. We are indebted to Amalia Montes and Lorenzo Seguido for their technical assistance, and to Drs. Maximiano Rodriguez-Lopez, Marfa Dolores Garcla and Hans Reichenbach for supplying the microorganisms.

This work was supported by a Fellowship from the Spanish Ministerio de Educaci6n y Ciencia, and by two contracts from the Spanish Division de Ciencias, CSIC.

281

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Distribution of adenosine 5′-triphosphate (ATP)-dependent hexose kinases in microorganisms