Methanococcus jannaschii Coenzyme F₄₂₀ Analogs Contain a Terminal α-Linked Glutamate

Abstract

Analyses of the F₄₂₀s present in Methanococcus jannaschii have shown that these cells contain a series of γ-glutamyl-linked F₄₂₀s capped with a single, terminal α-linked l-glutamate. The predominant form of F₄₂₀ was designated as α-F₄₂₀-3 and represented 86% of the F₄₂₀s in these cells. Analyses of Methanosarcina thermophila, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, and Mycobacterium smegmatis showed that they contained only γ-glutamyl-linked F₄₂₀s.

Coenzyme F₄₂₀ (Fig. 1) is a name given to a group of redox active cofactors that are presently known to have only a limited distribution among the archaea and high G+C gram-positive bacteria (3). Although playing a crucial role in methanoarchaeal metabolism (4), coenzyme F₄₂₀ has also been found in various eubacteria such as Streptomyces, Rhodococcus, Nocardioides, and Mycobacterium spp. and their relatives (3). The coenzyme, in fact, was first isolated in 1960 as a cofactor involved in the biosynthesis of chlortetracycline in Streptomyces aureofaciens (18). The coenzyme is presently known to be involved in the biosynthesis of a number of secondary metabolites (20), the degradation of nitroaromatics (5), and activation of nitroimidazofurans (23). Mycobacterium and Nocardia spp. contain an F₄₂₀-dependent glucose-6-phosphate dehydrogenase (21, 22), and an F₄₂₀ containing photolyase functions in DNA repair mechanisms in a number of microorganisms (7).

FIG. 1.

The chemical structures of the F₄₂₀ coenzyme analogs and their biosynthetic precursors. FO is defined in the text and is the fluorescent chromophore in F₄₂₀.

The first F₄₂₀ whose structure was completely characterized was the γ-F₄₂₀-2 [N-(N-l-lactyl-γ-l-glutamyl)-l-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin] isolated from Methanobacterium thermoautotrophicum (γ-F₄₂₀-2 in Fig. 1) (6). This F₄₂₀ contained two glutamic acids, with the terminal glutamate being bound through an amide bond to the γ position of the other glutamate, as is found in most folates and glutathione. This structure will be designated here as γ-F₄₂₀-2 to indicate the γ attachment of this terminal glutamate to the core structure of F₄₂₀-1 (N-l-lactyl-γ-l-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin) (Fig. 1). Subsequent work, however, has revealed that most organisms contain a series of γ-polyglutamated F₄₂₀ cofactors with up to a total of seven γ-linked glutamates (1, 10-12, 17, 19).

The assembly of F₄₂₀-0 and its polyglutamate derivatives from 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) (Fig. 1), pyruvate, and glutamate requires at least six steps (14, 15). The fourth step in this sequence of reactions is the reaction of lactyl (2) diphospho-(5′) guanosine (LPPG) with FO to form F₄₂₀-0 (F₄₂₀ with no glutamic acid) and GMP. The glutamic acid is then added to the lactyl carboxyl group of F₄₂₀-0 (Fig. 1) to generate the F₄₂₀-1, which is followed by the repeated addition of single glutamates to generate the polyglutamate derivatives.

As part of our work to establish the genes and pathway involved in F₄₂₀ biosynthesis in Methanococcus jannaschii (13-15) we examined the F₄₂₀ species present in this euryarchaeon. These analyses established that the F₄₂₀s present in these cells are unique and consist of a series of γ-linked F₄₂₀s capped with a single terminal α-linked l-glutamate.

A cell extract of M. jannaschii was prepared by sonication of 4.67 g of frozen cells suspended in 10 ml of TES extraction buffer {50 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid]-NaOH-10 mM MgCl₂ [pH 7.5]} under Ar for 5 min at 3°C. The resulting mixture was centrifuged under Ar (10 min at 27,000 × g) and stored frozen at −20°C until used. The protein concentration of the M. jannaschii extract was 38 mg/ml. Protein concentrations were measured using the BCA total protein assay (Pierce, Rockford, Ill.) with bovine serum albumin as a standard. Similar procedures were used to prepare cell extracts of Methanosarcina thermophila, Methanosarcina barkeri, Methanobacterium thermoautotrophicum ΔH, and Archaeoglobus fulgidus, having protein concentrations of 38, 6.8, 37, and 20 mg/ml, respectively. Mycobacterium smegmatis cell pellet (104 mg) was extracted by heating for 10 min at 100°C with 0.5 ml of water. After the addition of 0.6 ml of methanol and after centrifugation (14,000 × g for 5 min) to remove insoluble material, the resulting clear yellow extract was separated, evaporated, and dissolved in water prior to high-performance liquid chromatography (HPLC) analysis.

F₄₂₀ present in the cell extracts produced by sonication was isolated by precipitation of the proteins from the cell extracts (50 μl) with the addition of 80 μl of methanol, followed by centrifugation (14,000 × g for 5 min). After separation from the pellet, the clear liquid was diluted to 1 ml with water and 20-μl portions were analyzed by HPLC. Analyses were performed on a Shimadzu SCL-6B HPLC using a C-18 reversed phase column (AXXI-Chrom octyldecyl silane column; 5-μm particle size; 4.6 mm [internal diameter] by 25 cm) eluted isocratically with 15% methanol in 25 mM sodium acetate (pH 6.5) buffer at a flow rate of 0.5 ml per min. The eluent was monitored by fluorescence (excitation wavelength, 420 nm; emission wavelength, 480 nm) and by absorbance at 280 nm. By use of this HPLC method, the different F₄₂₀ derivatives showed the following retention times: γ-F₄₂₀-7, 4.88 min; γ-F₄₂₀-6, 5.04 min; γ-F₄₂₀-5, 5.84 min; γ-F₄₂₀-4, 5.99 min; α-F₄₂₀-5, 6.05 min; α-F₄₂₀-4, 6.51 min; γ-F₄₂₀-3, 6.95 min; α-F₄₂₀-3, 7.41 min; γ-F₄₂₀-2, 9.07 min; F₄₂₀-1, 13.0 min; F₄₂₀-0, 26.5 min; FO-P, 27.8 min; and FO, 30.4 min. The γ-F₄₂₀s were identified by coinjection with previously characterized γ-F₄₂₀ samples from Methanosarcina barkeri and Mycobacterium smegmatis (1). Those peaks not corresponding with these known samples were the α-F₄₂₀s and were identified with regard to the number of glutamates by their elution positions relative to the known samples and by the observed difference between the elution times of F₄₂₀s that differed by one glutamate.

HPLC analysis of the F₄₂₀s from M. jannaschii showed that they consisted of four major fluorescent peaks (percent of total fluorescence): γ-F₄₂₀-2 (2.9%), α-F₄₂₀-3 (85.9%), α-F₄₂₀-4 (10.1%), and α-F₄₂₀-5 (1.5%) (Table 1). The measured total amount of F₄₂₀ in this organism (>2.0 μmol/g [dry weight]) was among the highest reported for any organism (16). Except for the γ-F₄₂₀-2 peak, each peak had chromatographic retention times different from that of the γ-linked F₄₂₀ present in any of the other archaea tested, which included Methanosarcina thermophila, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, and Mycobacterium smegmatis. These organisms were found to have the distributions of γ-F₄₂₀ (percent of total fluorescence) shown in Table 1. No F₄₂₀-1 was detected in any sample.

TABLE 1.

Distribution and types of F₄₂₀ in the assayed cells

F420	Distribution of F420 type (% of total F420s) in:
	Methano- coccus jannaschii	Methano- sarcina thermophila	Methano- sarcina barkeri	Archaeo- globus fulgidus	Myco- bacterium smegmatis
γ-F420-2	2.9	2.4	31	0.3	Trace
γ-F420-3		19	6.8	0.5
α-F420-3	85.9
γ-F420-4		62	32	89a
α-F420-4	10.1
γ-F420-5		16	25		67
α-F420-5	1.5
γ-F420-6		1.0	4.0	10b	31
γ-F420-7		Trace	0.7		7.2

^aThe sum of γ-F₄₂₀-4 and -5 was 89%.

^bThe sum of γ-F₄₂₀-6 and -7 was 10%.

To establish the nature of the linkages in the samples of F₄₂₀, they were incubated with peptidases of known specificity and the products were measured by HPLC. The enzymes used were carboxypeptidase Y (a peptidyl-l-amino acid hydrolase that specifically removes carboxyl-terminal α-amino acids) and glutamyltranspeptidase and carboxypeptidase G (both γ-glutamyl hydrolases that specifically cleave terminal γ-linked glutamyl peptide bonds). Thus, treatment of 2 μl of M. jannaschii cell extract with 10 μl of a solution of carboxypeptidase Y from baker's yeast (Sigma) (4.35 U in 50 mM TES-Na⁺-10 mM MgCl₂ [pH 7.5]) for 1 h at room temperature converted the sample to a mixture of γ-F₄₂₀-2, γ-F₄₂₀-3, and γ-F₄₂₀-4 based on their HPLC retention times. The abundances of F₄₂₀s in the resulting mixture matched the ratios of the starting F₄₂₀s. Treatment of the extracts containing the F₄₂₀ analogs from the other organisms in the same manner had no effect on their HPLC profiles. Treatment of all of the cell extracts with glutamyltranspeptidase type IV from porcine kidney (Sigma) as previously described (15) had no effect on the M. jannaschii α-F₄₂₀s but degraded all of the other γ-F₄₂₀s, including the γ-F₄₂₀-2 present in M. jannaschii, to F₄₂₀-1. Treatment of 2 μl of M. jannaschii cell extract with 10 μl of carboxypeptidase G from Pseudomonas spp. (Sigma) (50 U per ml of 50 mM TES-Na⁺-10 mM MgCl₂ [pH 7.5]) for 2 h at room temperature had no effect on the major M. jannaschii α-F₄₂₀s but did hydrolyze the γ-F₄₂₀-2 present in M. jannaschii to F₄₂₀-1. A similar treatment of the F₄₂₀ samples from the other organisms degraded all of them to F₄₂₀-0.

For the purification of the F₄₂₀ analogs from M. jannaschii, cell extracts were separated on a MonoQ HR 5/5 column on a BioLogic HR chromatographic system (Bio-Rad) using a linear sodium chloride gradient. Buffer A was 25 mM Tris-HCl [pH 7.5] buffer, and buffer B was the same but containing 1 M NaCl. The total flow was 0.5 ml per min for 40 min. In this system, the F₄₂₀ eluted at 0.6 M NaCl and was identified by its fluorescence intensity (excitation, 420 nm; emission, 480 nm). The F₄₂₀-containing fractions were combined and concentrated and applied to a C-18 column (0.5 by 10 cm; 55-105 μm; Waters) equilibrated with water. The F₄₂₀ was eluted with 10% methanol in water. The F₄₂₀-containing fractions were combined and concentrated, formic acid was added to a concentration of 1%, and the sample was applied to a C-18 column (0.5 by 10 cm; 55-105 μm; Waters) equilibrated with 1% formic acid. The F₄₂₀, which was tightly bound to the column, was then eluted with a step gradient of 1% formic acid-methanol. The F₄₂₀ was eluted around 50% methanol. The F₄₂₀, which has no color at this pH, was observed via its fluorescence under a UV light. The final material was then purified by preparative thin-layer chromatography using the solvent system acetonitrile-water-formic acid (80:20:10 [vol/vol/vol]) with a R_f of 0.22. This material was subjected to acid hydrolysis (6 M HCl; 110°C; 12 h), and the resulting amino acids were converted into their N-trifluoroacetyl methyl ester derivatives for gas chromatography-mass spectrometry (GC-MS) analysis. GC-MS analysis as previously described (25) showed only the presence of the glutamate derivative. GC-MS analysis of the sample using an Alpha Dex 120 fused chiral silica capillary column (30 m by 0.25 mm by 0.25-μm film thickness; Supelco, Bellefonte, Pa.) showed that it was composed of only l-glutamic acid.

In total, these data are consistent with the conclusion that the F₄₂₀s in M. jannaschii, consisting of γ-F₄₂₀-2, γ-F₄₂₀-3, and γ-F₄₂₀-4, each have a single α-linked terminal glutamate, as shown in Fig. 1. These structures will be referred to as α-F₄₂₀. The cells also contain a small amount of γ-F₄₂₀-2, which could serve as the biosynthetic precursor to the other structures.

Although no example of α-linked glutamates in F₄₂₀ has been described in any organism, the occurrence of α-linked glutamates in folates in E. coli (8) and an α-glutamylmethanopterin, sarcinapterin, in Methanosarcina and some Methanococcus species has been described (24). In the case of the folates, it has been established that a bifunctional dihydrofolate synthetase-folylpolyglutamate synthetase (FolC) adds the first glutamate to pteroic acid and then up to three additional γ-linked glutamates. A second enzyme then extends the folylpolyglutamate chain via the addition of α-linked glutamates (9). The enzyme adding these α-linked glutamates was purified and shown to differ from FolC, but the gene for its generation was not identified. It is very likely that the same sequence of events is occurring in M. jannaschii. In M. jannaschii, there could be one enzyme, which would add the glutamate to F₄₂₀-0 to generate the series of γ-linked glutamate F₄₂₀ analogs. This process would be analogous to that with the bifunctional FolC. The resulting γ-linked glutamate F₄₂₀ analogs would then serve as substrates for the addition of the α-linked glutamate. In each case, the addition of the glutamates would likely proceed via acylphosphate intermediates, generated from a nucleotide triphosphate, as is known to occur with FolC (2). The major difference however, between the biosynthesis of the α-linked glutamates in folates and the M. jannaschii F₄₂₀s is that only one α-linked glutamate would be added.

Since M. jannaschii contains α-linked glutamate F₄₂₀ analogs, many enzymes in these cells which are known to use the γ-linked glutamate F₄₂₀ analogs in other bacteria and archaea must have adapted in M. jannaschii to use the α-linked analogs. This list of enzymes for M. jannaschii in which F₄₂₀ is essential would include hydrogenase, formate dehydrogenase, methylene-tetrahydromethanopterin dehydrogenase, alcohol dehydrogenase, methylene-tetrahydromethanopterin reductase, and F₄₂₀-dependent NADP⁺ oxidoreductase. Questions still to be answered are how and why this specific change in coenzyme structure specifically occurred in M. jannaschii and what enzymes are involved.