VARIANT CODES for N-acetylglucosamine (NAG) utilization:

1 NAG core enzymes (nagA+nagB) + NAG PTS uptake systems (nagE)
2 NAG core enzymes (nagA+nagB) + NAG kinase (either one variant of nagK)
3 NAG core enzymes (nagA+nagB). No NAG kinase. No NAG PTS. Might be involved in sialic acid utilization, which merge with NAG utilization at NAG-P.

1X or 2X the same as 1 and 2 but one of the two core enzymes (either nagA or nagB) is absent.

-1 No NAG utilization

Notes copied from N-Acetyl-D-Glucosamine_Utilization:
This project is now joined by D.Rodionov.
It is very much project in progress.
Take everything with a pound of salt (AO)
For example NagC-NAGL prediction is very shaky and may be wrong.
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AO:
Additions about Shewanella (in collaboration and coordination with Margie Romine)
Prediction:
NAGP: N-Acetyl-D-glucosamine permease, possible
in Shewanella
Inferred based on chromosomal clustering in Shewanella ssp.
It is supported by the fact that Shewanella doesn't seem to have NagE like dedicated PTS system (at least not in the cluster). It also does not seem to have ABC-system as in E.coli

NAGX: N-Acetyl-D-glucosamine utilization related protein, possible

Can it be NAG kinase in Xanthomonas, Xyllella?
fig|160492.1.peg.1456: Xylella fastidiosa 9a5c
NagKII: N-acetylglucosamine kinase bacterial type 2, possible (EC 2.7.1.59)
(That woudl be yet another version of NAG kinase! Seems like a pretty standard thing with these kinases. Look at PanK as another example)

NAGR:
Transcriptional regulator, LacI family, possibly related to NAG
inferred by clustering in gammaproteobacteria. Coexists with GntR in some.
Unclear (experimental).

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This subsystem is a joint project of OlgaZ + AndreiO (= Love and Piece)
NEW PROBLEM: how to tell apart glucosamine (nag operon) and galactosamine (aga operon) related enzymes

New role - ANAG: Involved in the degradation of heparan sulfate in human and other mammals. Hydrolysis of terminal non-reducing N-acetyl-D-glucosamine residues in N-acetyl-alpha-D-glucosaminides.

Subsystem: N-Acetyl-D-Glucosamine Utilization
(in development, advanced stage)
Salvage and recycling of N-Acetyl-D-Glucosamine (GlcNAc), a major component of complex carbohydrates, is present in a variety of forms in many species. In mammals, GlcNAc salvage merges with de novo biosynthesis (see below) of UDP-GlcNAc, a key precursor in the synthesis of sialic acid, N/O-glycan conjugates and protein O-glycosylation. De novo biosynthesis of UDP-GlcNAc also plays a crucial role in the biogenesis of bacterial cell wall biogenesis. This pathway is conserved in most bacteria and it is represented by a separate SEED subsystem, “UDP-N-acetylmuramate from Fructose-6-phosphate Biosynthesis.” It is related but distinct from the mammalian pathway (by the relative order of acetylation and phopshomutase reactions).

This difference may be one of the reasons why bacterial catabolism of GlcNAc proceeds differently. Unlike, mammals, most bacteria don’t seem to have Phosphoacetylglucosamine mutase (EC 5.4.2.3, PAGAM)*. Therefore, GlcNAc-6P undergoes deacetylation and further catabolism to F6P, instead of direct conversion to GlcNAc-1P, required for the synthesis of UDP-GlcNAc (see pathway diagrams and KEGG map: Aminosugars metabolism).
====
* footnote. Although some bacterial enzymes in KEGG are shown as PAGAM, they are likely misannotation. At least according to our other subsystem they are PGAMs variant (GlcN-P mutases)

In the current subsystem “N-Acetyl-D-Glucosamine Utilization” we are focusing on the main theme characteristic of bacteria: catabolism of exogenous or endogenous GlcNAc to F6P, while only indicating branching reactions (such as NAMPE and NAGPM, see diagram).
A signature of this subsystem (the most ubiquitous, conserved and committed enzyme) is N-acetylglucosamine-6-phosphate deacetylase (EC 3.5.1.25, NAGPD). Its presence in the genome pointe to a possibility of finding other components.

********Step-by-step commentaries:****************
1. Source of GlcNAc
A starting point metabolite GlcNAc may enter the catabolism form a variety of sources. Currently only one of the hydrolytic enzymes is shown (Beta-hexosaminidase (EC 3.2.1.52) ,BHA), since a respective gene is often clustered with other genes of GlcNAc catabolism (so called nag operon in E.coli [eg REF 1]). Other oligosaccharides and hexosaminidases may be added as appropriate.

A critical entry point into catabolism/salvage/recycling is phosporylation to GlcNAc-6P. This step is often directly coupled to transport of exogenous GlcNAc. At least two variants of GlcNAc uptake may be identified; Variant #1 is a well-known PTS system (coupled with PEP-dependent phosphorylation by IIABC component (EC 2.7.1.69)) and hypothesized Variant #2 ABC transporter system (directly coupled with post-uptake kinase reaction by N-acetylglucosamine kinase (EC 2.7.1.59)).

Variant #1 is described in E.coli [REF 1], and GlcNAc-specific IIABC component is part of nag operon. Although, it is always difficult to unambiguously identify a specificity of PTS components, in this subsystem we did using clustering with NAG signature genes and very close similarity. It is likely that PTS components supporting GlcNAc uptake in many other species remain unidentified. Some of them may have a broader specificity towards other sugars (hence, other sugar-specific PTS components may be always considered as candidates for the GlcNAc uptake). Two other components of a standard PTS system: Phosphoenolpyruvate-protein phosphotransferase of PTS system (EC 2.7.3.9, PTS_PPT) and Phosphocarrier protein of PTS system (PTS_PCP) are less committed to a paricular metabolite. Their inclusion in the subsystem is not mandatory (we have mapped only some of them, particularly those that clustered with PTS_NAG.


Variant #2 (ABC transport) apparently is not present in E.coli and related bacteria. It may be described in the literature, but we haven’t specifically searched for the evidence. However, the analysis of chromosomal clustering (originally performed by OlgaZ in the case of T.maritima) allows to reliably predict an ABC transport system responsible for GlcNAc uptake. This prediction is very strong and it can be projected to a number of other species that lack a respective PTS, especially when supported by clustering (see a subsystem, examples of Brucella, Rhizobia, etc).

2. Phosphorylation of GlcNAc, a first committed step of catabolism/salvage

Most importantly, the absence of PTS combined with the requirement of GlcNAc phosphorylation, allowed us to reliably predict NagC-homologs present in many bacteria (including T.maritima) as the best candidate for an alternative (bacterial) form of N-acetylglucosamine kinase bacterial type predicted (EC 2.7.1.59, NAGKb). This prediction initially made based on the chromosomal clustering/subsystem analysis in T.maritima was posted on Subsystem Forum. This activity was detected in E.coli, but the respective gene was never found. This is most likely due a functional redundance of NAGK in E.coli, which has a PTS system. The absence of NAGK gene led to a speculation that in E.coli, catabolism of endogenous GlcNAc may proceed via excretion, followe by PTS-driven uptake and phosphorylation [REF 2]. If our prediction is correct (experiments are in progress in Osterman’s Lab), this cumbersome route may be replaced by a more straightforward internal utilization.

NAGK story. Although phosphorylation is the first committed step in GlcNAc catabolism, NAGK genes remained elusive for a long time. The first experimental identification of the eukaryotic form of NAGK was performed for the mouse and fungal enzymes [REF 3,4]. We now project this form over a number of bacterial species. This projection has to be done carefully, since the enzyme belongs to a large family with multiple paralogs of different specificity towards a variety of sugars. We used two additional criteria for such a projection:
- Genome context: clustering with the other nag genes.
- Functional context: need of NAGK and the absence of an alternative forms.
Based on this analysis, all cyanobacteria have a “eukaryotic form” of NAGK.

Prediction of NagC (commonly known as GlcNAc operon transcriptional regulator [REF 2]), as a non-orthologous (bacterial) form of NAGK is strongly supported by:
- genome context – clustering in T.maritima
- functional context – requirement for NAGK in the absence of PTS.
- Homology context – this protein belongs to a family of ROK kinases (such as bacterial glucokinases)
(NB: we may expect that at least in some organisms, such as some of the Staphylococci and all Streptococci, the same enzyme will display both activities for Glc and GlcNAc. We have not reannotated the resepctive genes in these organisms, and NAGK appears as a missing gene in some of them. Nevertheless, those proteins currently assigned as glucokinases are the prime candidates for NAGKb. In contrast, no good candidates of NAGKe form are present in these organisms. Focused experiments are required to resolve this conundrum). Another complexity associated with NagC is a presence of multiple paralogs (at least in some species). We attempted to annotate only those that are either clustered or are very close by sequence to them and termed some others: NAGK (or NagC) homologs. We also retained a “transcriptional regulator’ as a second function, although it is hard to tell whether this function is actually implemented in all of the organisms (beyond E.coli and close relatives). These questions also should be addressed experimentally.

3. Deacetylation of GlcNAc-6P, a signature step in bacteria

As already mentioned, NAGPD is the most conserved enzyme of the subsystem. Orthologs can be easily identified in mammals and other eukaryotes. Not much to be said about it other than the assignments are rather straightforward, besides it is very often clustered with other genes in the pathway. We used “pinning” with NAGPD as a way to deconvolute many other assignments related to the pathway.

4. Deamination-isomerization of GlcN-6P to F6P.

This is the last committed step of the bacterial pathway. Interestingly, glucosamine-6-phosphate deaminase G6PD, (EC 3.5.99.6) also seems to occur in two “flavors”, as a result of a nonorthologous gene displacement (NOG). The well-known form (NagB-like) was experimentally characterized in E.coli. It is a part of nag operon and it is often clustered with nag genes in other species.

A relatively new (predicted) form, G6Pda, was inferred initially by the analysis of nag-cluster in T.maritima (by OlgaZ as reflected in the initial Forum posting and in SEED annotation). TM0813 and its homologs in other species were predicted to have a Glucosamine-6-phosphate deaminase/isomerase activity, eg to operate only in one direction: from GlcNAc-6P to F6P. These proteins are the close homologs of the C-terminal domain of the GlmS (Glucosamine--fructose-6-phosphate aminotransferase [isomerizing] (EC 2.6.1.16), REF 5,6) family (encoded by another gene in T.maritima TM0148, which is also conserved in most bacteria). They lack the Gln-utilization (N-terminal) domain of GlmS-like enzymes, and hence they can not work in the biosynthetic pathway (from F6P via GlcAm-6P to UDP-NAc-muramate). On the other hand, the analysis of T. maritima cluster of genes (including TM0813) suggests its involvement with the NAcGlcAm catabolism, which includes the reverse reaction from GlcAm-6P F6P. Importantly (for this prediction), T.maritima does not contain NagB homologs, making TM0813 the best candidate for a function inferred by a subsystem (pathway) analysis.
This family is not homologous to NagB-like deaminases. The structure of this single-domain protein (TM0813) was solved at JCSG. The occurrence profile of G6PD and G6PDa represents an almost perfect anticorrelation, which also supports this prediction.

5. Branching-out reactions.

N-acetylmannosamine-6-phosphate 2-epimerase (EC 5.1.3.9). interconverting GlcNAc6P and ManNAc6P is a link between these aminosugars and N-Acetyneuraminate Metabolism

Phosphoacetylglucosamine mutase (EC 5.4.2.3) is a signature of eukarytic version of “UDP-N-acetylmuramate from Fructose-6-phosphate Biosynthesis”. This is an entry point for salvage of GlcNAc in mammals.

*********REFERENCES:***************

1: Plumbridge J, Vimr E.
Convergent pathways for utilization of the amino sugars N-acetylglucosamine,
N-acetylmannosamine, and N-acetylneuraminic acid by Escherichia coli.
J Bacteriol. 1999 Jan;181(1):47-54. PMID: 9864311

2: Plumbridge J, Pellegrini O. Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP.
Mol Microbiol. 2004 Apr;52(2):437-49. PMID: 15066032

3: Yamada-Okabe T, Sakamori Y, Mio T, Yamada-Okabe H.
Identification and characterization of the genes for N-acetylglucosamine kinase
and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida
albicans. Eur J Biochem. 2001 Apr;268(8):2498-505.
PMID: 11298769

4: Hinderlich S, Berger M, Schwarzkopf M, Effertz K, Reutter W.
Molecular cloning and characterization of murine and human N-acetylglucosamine
kinase. Eur J Biochem. 2000 Jun;267(11):3301-8.
PMID: 10824116 [PubMed - indexed for MEDLINE]

5: Obmolova G, Badet-Denisot MA, Badet B, Teplyakov A.
Crystallization and preliminary X-ray analysis of the two domains of
glucosamine-6-phosphate synthase from Escherichia coli.
J Mol Biol. 1994 Oct 7;242(5):703-5.
PMID: 7932726 [PubMed - indexed for MEDLINE]

6: Teplyakov A, Obmolova G, Badet B, Badet-Denisot MA.
Channeling of ammonia in glucosamine-6-phosphate synthase.
J Mol Biol. 2001 Nov 9;313(5):1093-102.
PMID: 11700065 [PubMed - indexed for MEDLINE]

7: Brinkkötter, H. KlöB, C.A. Alpert and J.W. Lengeler, Pathways for the utilization of N-acetyl-galactosamine and galactosamine in Escherichia coli. Mol. Microbiol. 37 (2000), pp. 125–135.

Additional references about PTS and ABC:

1: Saito A et al. Mutational analysis of the bi...[PMID: 15148605]
2: Dahl U et al. Identification of a phosphotr...[PMID: 15060041]
3: Alice AF et al. Phosphoenolpyruvate phosphotr...[PMID: 12855720]
4: Wang F et al. Streptomyces olivaceoviridis ...[PMID: 12436256]


********PROBLEMS:
Interestingly NAGK presence is a variable feature among staphs!


*********PREDICTIONS
Summary:
Firm:
1. NagC-homologs is an alternative NAGK;
2. ABC-transporters family involved in GlcNAc uptake (likely already known: see additional ref.1);
3. Single-domain glmS homologs (eg TM0813) is an alternative G6PD;

Fuzzy:
4. nagD may be a GlcNAc-P phosphatase or a phosphatase involved with PTS system.