[69a] Hexokinase

Joshi, Mira D.; Jagannathan, V.

doi:10.1016/0076-6879(66)09076-1

Cited by 55 publications

(23 citation statements)

References 4 publications

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“…1 . I .42) as described by Kelly & Hynes (1982); NAD-isocitrate dehydrogenase (EC 1.1.1.41) as described by Osmani & Scrutton (1983); NADP-malate dehydrogenase (decarboxylating) (EC 1.1.1.40) as described by McCullough & Roberts (1974); NADP-mannitol dehydrogenase (EC 1.1.1.67) as described by Niehaus & Dilts (1982) but using 200 mM-mannitol; hexokinase (EC 2.7.1.1) as described by Joshi & Jagannathan (1966); mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) as described by Hankinson & Cove (1975); citrate synthetase (EC 4.1.3.7) as described by Srere (1969); and mannitol-1-phosphate phosphatase (EC 3.1.3.22) as described by Hult & Gatenbeck (1979) but using 2 mM-mannitol-1-phosphate. The specificity of this latter enzyme assay was established by measurement of the rate of phosphate production from fructose 6-phosphate and sorbitol 6-phosphate under identical conditions.…”

Section: Methodsmentioning

confidence: 99%

NADPH Generation in Aspergillus nidulans: Is the Mannitol Cycle Involved?

Singh

Scrutton²,

Scrutton³

1988

Microbiology

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A cyclic pathway of NADPH generation involving interconversion of mannitol and fructose has been proposed to occur in fungi. In Aspergillus nidulans three enzymes of this proposed mannitol cycle (hexokinase, NADP-mannitol dehydrogenase and mannitol-1-phosphate phosphatase) were shown to be localized exclusively in the cytosol. Two isoenzymes of the fourth enzyme (mannitol-1-phosphate dehydrogenase) were detected and shown to be localized respectively in the mitochondrion and the cytosol. The mitochondrial isoenzyme appeared to be present on the outer face of the inner mitochondrial membrane. No evidence was found for a coordinated change in the maximal activities of the enzymes of the proposed mannitol cycle in extracts prepared from mycelia grown on six different carbon, and three different nitrogen sources nor for any increase in these activities induced by growth on NO;. Studies of this type in which other NADP-linked dehydrogenases were measured showed that for most carbon sources tested growth on NO: increased the maximal activity of NADP-isocitrate dehydrogenase as well as that of glucose-6-phosphate and 6-phosphogluconate dehydrogenases but had little effect on the maximal activity of NADP-malate dehydrogenase (decarboxylating). Our studies provide no support for the operation of the mannitol cycle, or for the proposed role of this cycle in NADPH generation in A . nidulans. INTRODUCTIONProvision of NADPH is a crucial requirement for the continued operation of anabolic pathways such as fatty acid synthesis (Walker & Woodbine, 1976), sterol synthesis (McCorkindale, 1976) and purine synthesis, which utilize this coenzyme as a source of reducing equivalents. NADPH is also used in fungi and other micro-organisms to make oxidized nitrogen sources, e.g. NO;, available for amino acid, purine and pyrimidine synthesis (Lehninger, 1975). In non-photosynthetic micro-organisms it is usually considered that NADPH is generated primarily by the oxidation of glucose 6-phosphate to ri bulose 5-phosphate using glucose-6-phosphate and 6-phosphogluconate dehydrogenases. However, oxidation of isocitrate by NADP-isocitrate dehydrogenase and of malate by NADP-malate dehydrogenase can also contribute to NADPH generation. More recently, Hult & Gatenbeck (1978) proposed a cyclic pathway (Fig. 1) in which interconversion of fructose and mannitol is linked to transfer of reducing equivalents from NADH to NADPH. The presence of the enzymes of the mannitol cycle has been shown in a number of Deuteromycetes (Hult et al., 1980) and evidence suggesting its operation as a mechanism for NADPH generation has been obtained in studies using Alternaria alternata (Hult & Gatenbeck, 1978). However, doubts have been expressed about the operation of this cyclic pathway (McCullough et al., 1986) based on the very unfavourable K , of mannitol dehydrogenase for mannitol (Niehaus & Dilts, 1982) and on the lack of a coordinated ~~~~ ~~~~ t Present address :

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Section: Methodsmentioning

confidence: 99%

NADPH Generation in Aspergillus nidulans: Is the Mannitol Cycle Involved?

Singh

Scrutton²,

Scrutton³

1988

Microbiology

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“…ATP citrate lyase was assayed by measuring the fall in absorbance a t 340 nm after coupling to malate dehydrogenase [20]. Hexokinase was determined by coupling the reaction t o glucose-6-phosphate dehydrogenase [21]. The presence of 6-phosphogluconate dehydrogenase was corrected for in the blank which did not contain ATP.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Relationship between Fatty‐Acid and Phenol Synthesis in Aspergillus fumigatus

Ward

Packter

1974

European Journal of Biochemistry

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Investigations have been conducted into the relationship between fatty acid and phenol synthesis in submerged cultures of Aspergillus fumigatus. Both groups of metabolites are derived from acetate but phenol synthesis only occurs a t a late stage in the incubation period and is dependent on a change in the internal environment of the mycelium. Secretion of acetate-derived phenols commenced many hours after exhaustion of ammonium ions from the culture medium, or transfer of mycelium to glucose solution. Lipid synthesis, however, was maintained a t this stage and continued into the phenol-secreting phase when sufficient glucose was present to provide the necessary substrates. The additional lipid formed was identified as triglyceride.The composition of fatty acids in the total lipids was very similar to that found in other , Aspergillus sp. and related Ascomycetes and consisted principally of C18:2 with c16:0, c 1 6 : 1 , C1s:o and Cmi. Addition of 4-fluorophenylalanine, but not cycloheximide, caused a rapid reduction in fatty acid synthetase activity and lipid synthesis, and abolished formation of orsellinic acid and trihydroxytoluene, the normal secreted products.Certain enzymes examined, including the dehydrogenases of the pentose phosphate pathway, maintained their specific activity during the "stationary" phase whereas fatty acid synthetase was present at greatly reduced activity. The activity of ATP citrate lyase was severely reduced to an undetectable level after approximately 40 h incubation; acetyl-CoA would therefore not be made available for fatty acid, phenol or ergosterol synthesis after this time.Initiation of phenol synthesis cannot be ascribed simply to diversion of substrates from fatty acid synthesis or to reduction in fatty acid synthetase activity.Fatty acids together with a large group of naturally-occurring phenols are formed by means of condensation reactions between acetyl-CoA and malonylCoA. The synthesis of the latter products, however, entails few if any reductive steps and the oxygen functions from the malonyl residues are therefore often retained as phenolic groups. A phenomenon that is common to the synthesis of fatty acids and phenols in fungi lies in their formation as a response to deprivation of an essentialnutrient with a resultant disturbance to the internal environment of the cell [l]. Growth of the organism then ceases and the mycelium enters a stage that resembles the stationary Abbreviation. POPOP, p-bis,2-(5-phenyloxazolyl)benTrivial Name. Ergosterol, 5,7,22-ergostatrien-3B-01. Enzymes.

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“…Glucose kinase activity was measured according to methods described previously (24). Briefly, B. fragilis cells were grown in SAMM broth plus specified additions until a culture A 600 of approximately 0.8.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the RokA and HexA Broad-Substrate-Specificity Hexokinases from Bacteroides fragilis and Their Role in Hexose and N -Acetylglucosamine Utilization

Brigham

Malamy

2005

J Bacteriol

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Bacteroides fragilis, a human gastrointestinal commensal and an opportunistic pathogen, utilizes simple and complex sugars and polysaccharides for growth in the large intestine and at sites of infection. Because B. fragilis lacks transport-linked sugar phosphorylation systems, cytoplasmic kinase(s) was expected to be required for the phosphorylation of hexoses and hexosamines. We have now identified two hexose kinases that are important for growth of B. fragilis on glucose, mannose, and other sugars. One kinase (RokA), a member of the ROK family of proteins, was found to be the sole kinase for activation of N-acetyl-D-glucosamine (NAG). The other kinase (HexA) is responsible for the majority of the glucose kinase activity in the cell, although a hexA deletion mutant strain was not defective for growth on any substrate tested. Deletion of both the rokA and hexA kinase genes resulted in inability of the cell to use glucose, mannose, NAG, and many other sugars. We purified RokA and determined its approximate molecular mass to be 36.5 kDa. The purified RokA protein was shown to phosphorylate several substrates, including glucose, NAG, and mannose, but not N-acetylmannosamine or N-acetylneuraminic acid. Phylogenetic analysis of RokA showed that it is most similar to kinases from the Cytophaga-Flavibacterium-Bacteroides group, while HexA was most similar to other bacterial hexokinases and eukaryotic hexokinases.The "nanaerobic" gram-negative organism Bacteroides fragilis (4) is capable of utilizing a wide variety of simple sugars and complex oligosaccharides as sources of carbon and energy for growth. This is a useful property for a bacterium that is both a gastrointestinal tract commensal and an opportunistic pathogen. In the large intestine, undigested dietary polysaccharides and host-derived glycoproteins, such as colonic mucin, can serve as growth substrates for B. fragilis (22). At sites of infection, the availability of host cell surface glycoproteins and glycolipids provides nutrients during the establishment phase and after abscess formation. As an example, the cell surface Lewis antigen contains galactose, mannose, N-acetyl-D-glucosamine (NAG), and N-acetylneuraminic acid (34, 46), all of which can serve as carbon and energy sources for B. fragilis. Godoy et al. (17) demonstrated that the B. fragilis neuraminidase (the product of the nanH gene) catalyzes the removal of terminal sialic acids from surface polysaccharides of CHO cells in monolayer cultures and in the rat granuloma pouch model system, both of which are glucose-limited growth conditions. Accordingly, ⌬nanH mutant strains are compromised for growth in these model systems. N-acetyl-D-glucosamine is an excellent carbon and energy source for B. fragilis and appears to be utilized more efficiently than glucose (8). Release of NAG from host glycoproteins and its transport into the cell can also influence anabolic cellular processes. NAG is a major component of the murein sacculus (21), and the ability to acquire or produce NAG affects cell wall product...

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[69a] Hexokinase

Cited by 55 publications

References 4 publications

NADPH Generation in Aspergillus nidulans: Is the Mannitol Cycle Involved?

NADPH Generation in Aspergillus nidulans: Is the Mannitol Cycle Involved?

Relationship between Fatty‐Acid and Phenol Synthesis in Aspergillus fumigatus

Characterization of the RokA and HexA Broad-Substrate-Specificity Hexokinases from Bacteroides fragilis and Their Role in Hexose and N -Acetylglucosamine Utilization

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