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Concluding remarks
Abbreviations used: Co/Fe-S protein, corrinoid/iron-sulfur protein;
coenzyme F
, a 8-hydroxy-5-deazaflavin derivative; CoM-S-S-HTP, the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate; HS-CoM, coenzyme M, 2-mercaptoethanesulfonic acid;H SPT, 5,6,7,8-tetrahydrosarcinapterin; MAP, methyltransferase activation protein; MT , methanol:5-hydroxybenzimidazolylcobamide methyltransferase; MT , Co-methyl-5-hydroxybenzimidazolylcobamide: HS-CoM methyltransferase; MTZ, metronidazole.
In this chapter three subjects are reviewed. First, the two notable differences in our study of the methanol:coenzyme M methyltransferasereaction as compared to the work of Van der Meijden et al. [15] arediscussed. Next, the activation mechanism of methanol:5-hydroxy-benzimidazolylcobamide methyltransferase (MT ) is evaluated. In the final section, this mechanism is compared with the activation mechan-isms of other methanogenic methyl group transfer-catalyzing enzymes.
8.1. Differences with the work of Van der Meijden et al.
In our hands four proteins are required for transfer of the methyl group of methanol to coenzyme M (HS-CoM). Two of thesecomponents are responsible for the overall transfer of the methylmoiety, viz. methanol:5-hydroxybenzimidazolylcobamide methyl-transferase (MT ) and Co-methyl-5-hydroxybenzimidazolylcobamide: HS-CoM methyltransferase (MT ). MT was purified before by Van der Meijden et al. to approximately 90% homogeneity with a purificationfactor of 2.8 on the basis of the specific activity and a purificationfactor of 5.7 on the basis of the B -HBI content per mg of protein [17]. They found 3.4 mol of B -HBI per mol of protein [17]. It is the corrinoid content that is not confirmed by us [Chapter 6]. We repeat-edly purified large quantities of MT to 100% homogeneity, with a purification factor of 33 (on the basis of the specific activity), fromvarious batches of cell extract and always obtained an amount of about1.7 (± 0.4) mol B per mol of protein. This is only half of the amount reported by Van der Meijden et al. [17]. Since they only purified MT1once to 90% purity, we believe that our (reproducible) data from the100% pure protein is much more reliable.
The amount of MT present in cell extract varied, depending on the batch used, between 2.9 and 10% of the total amount of protein [P.
Daas, unpublished results]. Van der Meijden et al. reported an amountof about 15% [17]. Perhaps these varying amounts of MT reflect small differences in growth phase of Methanosarcina barkeri during har-vesting of the cells. This could point to some regulatory mechanism ofMT synthesis during growth. All in all, the body of the work of Van der Meijden et al. corroborated well with our results.
8.2. Reductive activation of MT1
MT is only able to accept the methyl group of methanol when the central cobalt atom of its corrinoids are in their fully reduced Co(I)state [16]. This so-called reductive activation results from the combinedaction of H , hydrogenase, Methyltransferase Activation Protein (MAP), and ATP [Chapter 2]. Ferredoxin is not absolutely required but itstimulates the apparent reaction rate of methyl group transfer [Chapter2]. These components activate MT by (i) producing reducing equival- ents for the actual reduction of the cobalt atom of the corrinoids (H , hydrogenase, ferredoxin) and by (ii) facilitating the reduction of thecob(II)amide of MT (MAP, ATP) [Chapter 6]. The action of the reducing system and the cob(II)amide modifying system will bediscussed below. All reactions involved in the transfer of the methylgroup from methanol to coenzyme M and the activation of MT are 8.2.1. The reducing system
H and hydrogenase are able to catalyze the one-electron reduction of the cob(III)amide and the "base-off" coordinated cob(II)amide of MT1to the "base-on" cob(II)amide and the cob(I)amide of MT , respectively [Chapter 6]. Ferredoxin enhances one or both of these reductions[Chapter 2]. In M. barkeri two distinct hydrogenases are present: ahigh-molecular-weight coenzyme F -dependent hydrogenase [2], and a non-F -dependent hydrogenase of lower molecular weight [9]. In the presence of the artificial electron acceptor metronidazole (MTZ) themethanol:HS-CoM methyltransferase reaction is almost completelyinhibited [4]. A maximum velocity of methyl transfer is establishedafter MTZ has been fully (and irreversibly) reduced. Under H -atmo- sphere, MTZ can not be reduced by hydrogenase directly, but specific-ally requires the mediation of ferredoxin or flavodoxin [1]. In view ofthe fact that MT does not contain iron-sulfur clusters [Chapter 6] reduction of MTZ must be catalyzed by the hydrogenase-ferredoxincouple. Since only the F ferredoxin [2,10], it is evident that this type of hydrogenase is able todonate electrons for the reduction of the cobalt atom of the corrinoidsof MT . A role for the non-F process seems unlikely [9,10], but can not be fully excluded as methyltransfer is not fully inhibited by MTZ [4] and activation of MT is possible in absence of ferredoxin [Chapter 2].
Fig. 1. Carbon and electron flow scheme proposed for transfer of the methyl group
from methanol to coenzyme M in Methanosarcina barkeri. The overall methyltrans-
ferase reaction is drawn in bold lines. The sequence of reactions involved in the
reactivation of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT )
is indicated by the thin lines. [Co(III)], [Co(II)], and [Co(I)] represent the variousoxidation states of the cobalt of the corrinoid prosthetic groups of MT .
Coordination of N-3 of the 5-hydroxybenzimidazolyl base is illustrated by theconnecting line between Co and N. Because the coordination of the base isunknown for the methylated corrinoids of MT a dashed line is drawn here. In the "base-off" Co(II) state no ligand is shown but it is possible that a ligand with poordelocalization properties, e.g. water, is present. The dashed line used for thedephosphorylation of MAP-phosphate indicates that the exact sequence of eventsfor this particular reaction is not fully established. BES, 2-bromoethanesulfonicacid; Fd, ferredoxin; HS-CoM, coenzyme M; Hyd, hydrogenase; MT , Co-methyl- 5-hydroxybenzimidazolylcobamide:HS-CoM methyltransferase. Ferredoxin is shownin parenthesis because activation can occur in absence of this protein.
8.2.2. The cob(II)amide modifying system
In absence of MAP or ATP reduction of the base-on coordinated Co(II) corrinoids of MT is impossible. Both components are required for the conversion of "base-on" into "base-off" cob(II)amides of MT1[Chapter 6]. In non-protein bound corrinoids the "base-off" form ismuch easier to reduce then the base-coordinated form [Chapter 4]. Inan identical way, it is conceivable that MAP and ATP enable thereduction of the cob(II)amide of MT by electrons derived from hydrogen. Since MAP interacts with ATP during the activation of MT1and is phosphorylated by the terminal phosphate of ATP [Chapter 5] itis suggested that phosphorylated MAP is the actual effector in theconversion of "base-on" to "base-off" Co(II) MT [Chapter 6]. Whether, the phosphate of phosphorylated MAP is actually transferred to MT1during this process is unknown. But it is evident that MAP must bedephosphorylated eventually, for it is isolated in a form which is fullyinactive in absence of ATP [Chapter 2].
8.3. Activation of methanogenic methyltransferases
When all results, known to date, with respect to the effect of ATP, MAP, and Ti(III)citrate on the methyl group transfer reactions catalyzedby M. barkeri and Methanobacterium thermoautotrophicum arecombined, an intriguing phenomenon is revealed. From the datapresented in Table 1 it is clear that there exist, at least, three differentmechanisms for the reductive activation of methyltransferases inmethanogens.
An activation mechanism which strictly requires ATP andinvolves the action of MAP. This system is involved in theactivation of MT and probably also in the activation of the enzymes concerned with methyl group transfer from tri-,di-, and monomethylamine to HS-CoM.
An activation mechanism which is completely independentof ATP. The activation of the corrinoid/iron-sulfur (Co/Fe-S) protein involved in methyl group transfer from acetyl-coenzyme A to tetrahydrosarcinapterin (H SPT) is the best Table 1. Influence of ATP, MAP, and Ti(III)citrate on methanogenic methyltransferase
Methanobacterium thermoautotrophicumc a Explanation of the signs used: +, stimulation; –, no effect; ?, unknown. Brackets are used to indicate results obtained from partially resolved systems.
b Includes unpublished results of Roel Wassenaar, with permission.
c Here CoM-S-S-HTP also stimulates the methyltransferase reaction [12]. Identical result were obtained for strain strain ∆H and Marburg. MAP is not involved because the proteinis not present in these organisms [P. Daas, unpublished results].
An activation mechanism which is stimulated by, but notabsolutely dependent on, ATP and does not involve theaction of MAP. Here, Ti(III)citrate is able to substitute forthe activation system. The membrane-bound methyl-H SPT: coenzyme M methyltransferase of M. barkeri and thecorresponding enzymes of M. thermoautotrophicum arerepresentatives of this group. It should be noted, however,that in the latter organism the heterodisulfide of HS-CoMand 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) also stimulates methyl group transfer [12] which isnot the case in M. barkeri [7]. The methanol:H SPT methyltransferase from M. barkeri [Chapter 7] also belongsto this group.
Since acetate grown M. barkeri is able to convert methanol as well [8], this organism is an example of an archaeon which containsmethyltransferases representing all of the three groups described above;viz. MT , the Co/Fe-S protein of carbon monoxide dehydrogenase, and methyl-H SPT:HS-CoM methyltransferase. The three distinguishable activation mechanisms can thus be collectively present in a singleorganism. Methanogens are still a surprising microbial group ! REFERENCES
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