Surveilling the Potential for Precision Medicine-driven PD-1/PD-L1-targeted Therapy in HNSCC.

Immunotherapy is becoming an accepted treatment modality for many patients with cancer and is now approved for use in platinum-refractory recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Despite these successes, a minority of patients with HNSCC receiving immunotherapy respond to treatment, and few undergo a complete response. Thus, there is a critical need to identify mechanisms regulating immune checkpoints in HNSCC such that one can predict who will benefit, and so novel combination strategies can be developed for non-responders. Here, we review the immunotherapy and molecular genetics literature to describe what is known about immune checkpoints in common genetic subsets of HNSCC. We highlight several highly recurrent genetic lesions that may serve as biomarkers or targets for combination immunotherapy in HNSCC.

Genomic sequencing and precision medicine in head and neck cancers.

Head and neck squamous cell carcinoma (HNSCC) remains a common and deadly disease. Historically, surgical and chemoradiation treatments have been met with modest success, and understanding of genetic drivers of HNSCC has been limited. With recent next generation sequencing studies focused on HNSCC, we are beginning to understand the genetic landscape of HNSCCs and are starting to identify and advance targeted options for patients. In this review, we describe current knowledge and recent advances in sequencing studies of HNSCC, discuss current limitations and future directions for further genomic analysis, and highlight the translational advances being undertaken to treat this important disease.

Crystal structure of the quorum-sensing protein LuxS reveals a catalytic metal site.

The ability of bacteria to regulate gene expression in response to changes in cell density is termed quorum sensing. This behavior involves the synthesis and recognition of extracellular, hormone-like compounds known as autoinducers. Here we report the structure of an autoinducer synthase, LuxS from Bacillus subtilis, at 1.6-A resolution (R(free) = 0.204; R(work) = 0.174). LuxS is a homodimeric enzyme with a novel fold that incorporates two identical tetrahedral metal-binding sites. This metal center is composed of a Zn(2+) atom coordinated by two histidines, a cysteine, and a solvent molecule, and is reminiscent of active sites found in several peptidases and amidases. Although the nature of the autoinducer synthesized by LuxS cannot be deduced from the crystal structure, features of the putative active site suggest that LuxS might catalyze hydrolytic, but not proteolytic, cleavage of a small substrate. Our analysis represents a test of structure-based functional assignment.

Folate activation and catalysis in methylenetetrahydrofolate reductase from Escherichia coli: roles for aspartate 120 and glutamate 28.

The flavoprotein Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate). The X-ray crystal structure of the enzyme has revealed the amino acids at the flavin active site that are likely to be relevant to catalysis. Here, we have focused on two conserved residues, Asp 120 and Glu 28. The presence of an acidic residue (Asp 120) near the N1-C2=O position of the flavin distinguishes MTHFR from all other known flavin oxidoreductases and suggests an important function for this residue in modulating the flavin reactivity. Modeling of the CH(3)-H(4)folate product into the enzyme active site also suggests roles for Asp 120 in binding of folate and in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. In the NADH-menadione oxidoreductase assay and in the isolated reductive half-reaction, the Asp120Asn mutant enzyme is reduced by NADH 30% more rapidly than the wild-type enzyme, which is consistent with a measured increase in the flavin midpoint potential. Compared to the wild-type enzyme, the mutant showed 150-fold decreased activity in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction and in the oxidative half-reaction involving CH(2)-H(4)folate, but the apparent K(d) for CH(2)-H(4)folate was relatively unchanged. Our results support a role for Asp 120 in catalysis of folate reduction and perhaps in stabilization of the 5-iminium cation. By analogy to thymidylate synthase, which also uses CH(2)-H(4)folate as a substrate, Glu 28 may serve directly or via water as a general acid catalyst to aid in 5-iminium cation formation. Consistent with this role, the Glu28Gln mutant was unable to catalyze the reduction of CH(2)-H(4)folate and was inactive in the physiological oxidoreductase reaction. The mutant enzyme was able to bind CH(3)-H(4)folate, but reduction of the FAD cofactor was not observed. In the NADH-menadione oxidoreductase assay, the mutant demonstrated a 240-fold decrease in activity.

Thioredoxin reductase two modes of catalysis have evolved.

Thioredoxin reductase (EC 1.6.4.5) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin plays several key roles in maintaining the redox environment of the cell. Like all members of the enzyme family that includes lipoamide dehydrogenase, glutathione reductase and mercuric reductase, thioredoxin reductase contains a redox active disulfide adjacent to the flavin ring. Evolution has produced two forms of thioredoxin reductase, a protein in prokaryotes, archaea and lower eukaryotes having a Mr of 35 000, and a protein in higher eukaryotes having a Mr of 55 000. Reducing equivalents are transferred from the apolar flavin binding site to the protein substrate by distinct mechanisms in the two forms of thioredoxin reductase. In the low Mr enzyme, interconversion between two conformations occurs twice in each catalytic cycle. After reduction of the disulfide by the flavin, the pyridine nucleotide domain must rotate with respect to the flavin domain in order to expose the nascent dithiol for reaction with thioredoxin; this motion repositions the pyridine ring adjacent to the flavin ring. In the high Mr enzyme, a third redox active group shuttles the reducing equivalent from the apolar active site to the protein surface. This group is a second redox active disulfide in thioredoxin reductase from Plasmodium falciparum and a selenenylsulfide in the mammalian enzyme. P. falciparum is the major causative agent of malaria and it is hoped that the chemical difference between the two high Mr forms may be exploited for drug design.

Interaction of flavodoxin with cobalamin-dependent methionine synthase.

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, forming tetrahydrofolate and methionine. The Escherichia coli enzyme, like its mammalian homologue, is occasionally inactivated by oxidation of the cofactor to cob(II)alamin. To return to the catalytic cycle, the cob(II)alamin forms of both the bacterial and mammalian enzymes must be reductively remethylated. Reduced flavodoxin donates an electron for this reaction in E. coli, and S-adenosylmethionine serves as the methyl donor. In humans, the electron is thought to be provided by methionine synthase reductase, a protein containing a domain with a significant degree of homology to flavodoxin. Because of this homology, studies of the interactions between E. coli flavodoxin and methionine synthase provide a model for the mammalian system. To characterize the binding interface between E. coli flavodoxin and methionine synthase, we have employed site-directed mutagenesis and chemical cross-linking using carbodiimide and N-hydroxysuccinimide. Glutamate 61 of flavodoxin is identified as a cross-linked residue, and lysine 959 of the C-terminal activation domain of methionine synthase is assigned as its partner. The mutation of lysine 959 to threonine results in a diminished level of cross-linking, but has only a small effect on the affinity of methionine synthase for flavodoxin. Identification of these cross-linked residues provides evidence in support of a docking model that will be useful in predicting the effects of mutations observed in mammalian homologues of E. coli flavodoxin and methionine synthase.

Twists in catalysis: alternating conformations of Escherichia coli thioredoxin reductase.

In thioredoxin reductase (TrxR) from Escherichia coli, cycles of reduction and reoxidation of the flavin adenine dinucleotide (FAD) cofactor depend on rate-limiting rearrangements of the FAD and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) domains. We describe the structure of the flavin-reducing conformation of E. coli TrxR at a resolution of 3.0 angstroms. The orientation of the two domains permits reduction of FAD by NADPH and oxidation of the enzyme dithiol by the protein substrate, thioredoxin. The alternate conformation, described by Kuriyan and co-workers, permits internal transfer of reducing equivalents from reduced FAD to the active-site disulfide. Comparison of these structures demonstrates that switching between the two conformations involves a "ball-and-socket" motion in which the pyridine nucleotide-binding domain rotates by 67 degrees.

[Human care: purification action]. / O cuidado humano: ação de purificação.

This article was originated by a request of some professors of the Masters Course in Nursing. The theme "bath", as an act of care, was developed, initially, on the meaning of moves. The esthetic of body movement, to the sound of music, was represented graphically, allowing to reach constructs as: relationship, physical contact, sharing, pleasure, satisfaction, cleanliness, reaproximation. The concept of "bath", understood through research in different bibliography and idioms, was translated as "purification". The meaning of "purification" was studied with an anthropological view. Many practices and rituals were perceived through history and human experience and bring with them tools that may help Nursing to create and recreate the experience and concept of human care. These elements and some contributions to comprehend care are shown in this study.

Crystal structure of reduced thioredoxin reductase from Escherichia coli: structural flexibility in the isoalloxazine ring of the flavin adenine dinucleotide cofactor.

Catalysis by thioredoxin reductase (TrxR) from Escherichia coli requires alternation between two domain arrangements. One of these conformations has been observed by X-ray crystallography (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816). This form of TrxR, denoted FO, permits the reaction of enzyme-bound reduced FAD with a redox-active disulfide on TrxR. As part of an investigation of conformational changes and intermediates in catalysis by TrxR, an X-ray structure of the FO form of TrxR with both the FAD and active site disulfide reduced has been determined. Reduction after crystallization resulted in significant local conformation changes. The isoalloxazine ring of the FAD cofactor, which is essentially planar in the oxidized enzyme, assumes a 34 degree "butterfly" bend about the N(5)-N(10) axis in reduced TrxR. Theoretical calculations reported by others predict ring bending of 15-28 degrees for reduced isoalloxazines protonated at N(1). The large bending in reduced TrxR is attributed in part to steric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by changes in the positions and interactions of several of the ribityl side-chain atoms of FAD. The bending angle in reduced TrxR is larger than that for any flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from those found in studies of free flavins, indicating that the protein environment has a significant effect on bending.

Refined structures of oxidized flavodoxin from Anacystis nidulans.

Flavodoxin from Anacystis nidulans (Synechococcus PCC 7942) was the first member of the flavodoxin family to be characterized, and is the structural prototype for the "long-chain" flavodoxins that have molecular masses of approximately 20 kDa. Crystal structure analyses and refinements of three orthorhombic forms of oxidized A. nidulans flavodoxin are reported, and salient features of the fold and the FMN binding site are compared with other flavodoxins. The structure of form I (wild-type: P212121, a=57.08 A, b=69.24 A, c=45.55 A), determined initially by multiple isomorphous replacement, has been refined to R=0.183 and R(free)=0.211 for data from 10.0 to 1.7 A resolution. Structures of form II (wild-type: P212121, a=60.05 A, b=65.85 A, c=51.36 A) and form III (Asn58Gly: P212121, a=51.30 A, b=59.15 A, c=94.44 A) have been determined by molecular replacement and refined versus data to 2.0 A and 1.85 A, respectively; the R values for forms II and III are 0.147 and 0.150. Changes in the molecular contacts that produce the alternative packings in these crystalline forms are analyzed. Deletion of the Asn side-chain in the mutant Asn58Gly removes an intermolecular stacking interaction and allows the alternative packing found in form III crystals. The functionally important 50's loop of the FMN binding site is less restrained by intermolecular contacts in these crystals but maintains the same conformation as in oxidized wild type protein. The structures reported here provide the starting point for structure-function studies of the reduced states and of mutants, described in the accompanying paper.

Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials.

The long-chain flavodoxins, with 169-176 residues, display oxidation-reduction potentials at pH 7 that vary from -50 to -260 mV for the oxidized/semiquinone (ox/sq) equilibrium and are -400 mV or lower for the semiquinone/hydroquinone (sq/hq) equilibrium. To examine the effects of protein interactions and conformation changes on FMN potentials in the long-chain flavodoxin from Anacystis nidulans (Synechococcus PCC 7942), we have determined crystal structures for the semiquinone and hydroquinone forms of the wild-type protein and for the mutant Asn58Gly, and have measured redox potentials and FMN association constants. A peptide near the flavin ring, Asn58-Val59, reorients when the FMN is reduced to the semiquinone form and adopts a conformation ("O-up") in which O 58 hydrogen bonds to the flavin N(5)H; this rearrangement is analogous to changes observed in the flavodoxins from Clostridium beijerinckii and Desulfovibrio vulgaris. On further reduction to the hydroquinone state, the Asn58-Val59 peptide in crystalline wild-type A. nidulans flavodoxin rotates away from the flavin to the "O-down" position characteristic of the oxidized structure. This reversion to the conformation found in the oxidized state is unusual and has not been observed in other flavodoxins. The Asn58Gly mutation, at the site which undergoes conformation changes when FMN is reduced, was expected to stabilize the O-up conformation found in the semiquinone oxidation state. This mutation raises the ox/sq potential by 46 mV to -175 mV and lowers the sq/hq potential by 26 mV to -468 mV. In the hydroquinone form of the Asn58Gly mutant the C-O 58 remains up and hydrogen bonded to N(5)H, as in the fully reduced flavodoxins from C. beijerinckii and D. vulgaris. The redox and structural properties of A. nidulans flavodoxin and the Asn58Gly mutant confirm the importance of interactions made by N(5) or N(5)H in determining potentials, and are consistent with earlier conclusions that conformational energies contribute to the observed potentials.The mutations Asp90Asn and Asp100Asn were designed to probe the effects of electrostatic interactions on the potentials of protein-bound flavin. Replacement of acidic by neutral residues at positions 90 and 100 does not perturb the structure, but has a substantial effect on the sq/hq equilibrium. This potential is increased by 25-41 mV, showing that electrostatic interaction between acidic residues and the flavin decreases the potential for conversion of the neutral semiquinone to the anionic hydroquinone. The potentials and the effects of mutations in A. nidulans flavodoxin are rationalized using a thermodynamic scheme developed for C. beijerinckii flavodoxin.

A prototypical cytidylyltransferase: CTP:glycerol-3-phosphate cytidylyltransferase from bacillus subtilis.

BACKGROUND: The formation of critical intermediates in the biosynthesis of lipids and complex carbohydrates is carried out by cytidylyltransferases, which utilize CTP to form activated CDP-alcohols or CMP-acid sugars plus inorganic pyrophosphate. Several cytidylyltransferases are related and constitute a conserved family of enzymes. The eukaryotic members of the family are complex enzymes with multiple regulatory regions or repeated catalytic domains, whereas the bacterial enzyme, CTP:glycerol-3-phosphate cytidylyltransferase (GCT), contains only the catalytic domain. Thus, GCT provides an excellent model for the study of catalysis by the eukaryotic cytidylyltransferases. RESULTS: The crystal structure of GCT from Bacillus subtilis has been determined by multiwavelength anomalous diffraction using a mercury derivative and refined to 2.0 A resolution (R(factor) 0.196; R(free) 0.255). GCT is a homodimer; each monomer comprises an alpha/beta fold with a central 3-2-1-4-5 parallel beta sheet. Additional helices and loops extending from the alpha/beta core form a bowl that binds substrates. CTP, bound at each active site of the homodimer, interacts with the conserved (14)HXGH and (113)RTXGISTT motifs. The dimer interface incorporates part of a third motif, (63)RYVDEVI, and includes hydrophobic residues adjoining the HXGH sequence. CONCLUSIONS: Structure superpositions relate GCT to the catalytic domains from class I aminoacyl-tRNA synthetases, and thus expand the tRNA synthetase family of folds to include the catalytic domains of the family of cytidylyltransferases. GCT and aminoacyl-tRNA synthetases catalyze analogous reactions, bind nucleotides in similar U-shaped conformations, and depend on histidines from analogous HXGH motifs for activity. The structural and other similarities support proposals that GCT, like the synthetases, catalyzes nucleotidyl transfer by stabilizing a pentavalent transition state at the alpha-phosphate of CTP.

The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia.

Elevated plasma homocysteine levels are associated with increased risk for cardiovascular disease and neural tube defects in humans. Folate treatment decreases homocysteine levels and dramatically reduces the incidence of neural tube defects. The flavoprotein methylenetetrahydrofolate reductase (MTHFR) is a likely target for these actions of folate. The most common genetic cause of mildly elevated plasma homocysteine in humans is the MTHFR polymorphism A222V (base change C677-->T). The X-ray analysis of E. coli MTHFR, reported here, provides a model for the catalytic domain that is shared by all MTHFRs. This domain is a beta8alpha8 barrel that binds FAD in a novel fashion. Ala 177, corresponding to Ala 222 in human MTHFR, is near the bottom of the barrel and distant from the FAD. The mutation A177V does not affect Km or k(cat) but instead increases the propensity for bacterial MTHFR to lose its essential flavin cofactor. Folate derivatives protect wild-type and mutant E. coli enzymes against flavin loss, and protect human MTHFR and the A222V mutant against thermal inactivation, suggesting a mechanism by which folate treatment reduces homocysteine levels.

A new decoration for nitric oxide synthase - a Zn(Cys)4 site.

Intense interest in the action and synthesis of nitric oxide has fueled structural studies of nitric oxide synthase (NOS). The monomeric and dimeric heme domains of inducible NOS were the first NOS structures to be described. A recent independent analysis of the corresponding heme domains from endothelial NOS confirms most of the features found earlier and also reveals a novel Zn(Cys)4 center - a new feature for NOS.

The mechanism of adenosylmethionine-dependent activation of methionine synthase: a rapid kinetic analysis of intermediates in reductive methylation of Cob(II)alamin enzyme.

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. During this primary turnover cycle, the enzyme alternates between the active methylcobalamin and cob(I)alamin forms of the enzyme. Formation of the cob(II)alamin prosthetic group by oxidation of cob(I)alamin or photolysis of methylcobalamin renders the enzyme inactive. Methionine synthase from E. coli catalyzes its own reactivation by a reductive methylation that involves electron transfer from reduced flavodoxin and methyl transfer from AdoMet. This process has been proposed to involve formation of a transient cob(I)alamin intermediate that is then trapped by methyl transfer from AdoMet. During aerobic growth of E. coli, electrons for this process are ultimately derived from NADPH, and electron transfer does not generate a detectable level of cob(I)alamin due to the large potential difference between the NADPH/NADP+ couple and the cob(I)alamin/cob(II)alamin couple. In this paper, we show that even in the presence of the strong reductant flavodoxin hydroquinone, cob(I)alamin is not observed as a significant intermediate. We demonstrate, however, that this is due to a rate-limiting reorganization of the cobalt ligand environment from five-coordinate to four-coordinate cob(II)alamin. Mutation of aspartate 757 to glutamate results in a cob(II)alamin enzyme that is approximately 70% four-coordinate, and reductive methylation of this enzyme using flavodoxin hydroquinone as the electron donor proceeds through a kinetically competent cob(I)alamin intermediate. Furthermore, wild-type cob(I)alamin enzyme produced by chemical reduction reacts with AdoMet in a kinetically competent reaction. We provide evidence that methyl transfer from AdoMet to cob(I)alamin enzyme results initially in formation of a five-coordinate methylcobalamin enzyme that slowly decays to the active six-coordinate methylcobalamin enzyme. We propose a kinetic scheme for reductive methylation of wild-type cob(II)alamin enzyme by adenosylmethionine and flavodoxin hydroquinone in which slow conformational changes mask the relatively fast electron and methyl transfer steps.

Emergence of epidemic O'nyong-nyong fever in Uganda after a 35-year absence: genetic characterization of the virus.

O'nyong-nyong (ONN) virus is an alphavirus (family Togaviridae, genus Alphavirus) classified in the Semliki Forest virus (SFV) antigenic complex. ONN was initially isolated in northern Uganda in 1959 during the early stages of an explosive arbovirus epidemic in which > 2 million cases were reported. No additional epidemics or human isolations of ONN were reported until 1996, when it was isolated from an epidemic in southern Uganda. We report the complete nucleotide and deduced amino acid sequence of one of these 1996-1997 ONN isolates (SG650) and that of the related alphavirus Igbo Ora virus. The data indicate that the recent ONN virus isolate is closely related to the previously published ONN strain isolated in 1959. In addition, phylogenetic analysis of the sequence data reveals that Igbo Ora virus, previously thought to be a separate virus closely related to ONN and Chikungunya (CHIK), clearly is a strain of ONN. The sequence data also reveal that unlike the published ONN (1959) sequence, all ONN strains from the 1996-1997 epidemic possess a stop codon at the nsp3-nsp4 junction.

Control of oxidation-reduction potentials in flavodoxin from Clostridium beijerinckii: the role of conformation changes.

X-ray analyses of wild-type and mutant flavodoxins from Clostridium beijerinckii show that the conformation of the peptide Gly57-Asp58, in a bend near the isoalloxazine ring of FMN, is correlated with the oxidation state of the FMN prosthetic group. The Gly-Asp peptide may adopt any of three conformations: trans O-up, in which the carbonyl oxygen of Gly57 (O57) points toward the flavin ring; trans O-down, in which O57 points away from the flavin; and cis O-down. Interconversions among these conformers that are linked to oxidation-reduction of the flavin can modulate the redox potentials of bound FMN. In the semiquinone and reduced forms of the protein, the Gly57-Asp58 peptide adopts the trans O-up conformation and accepts a hydrogen bond from the flavin N5H [Smith, W. W., Burnett, R. M., Darling, G. D., & Ludwig, M. L. (1977) J. Mol. Biol. 117, 195-225; Ludwig, M. L., & Luschinsky, C. L. (1992) in Chemistry and Biochemistry of Flavoenzymes III (Müller, F., Ed.) pp 427-466, CRC Press, Boca Raton, FL]. Analyses reported in this paper confirm that, in crystals of wild-type oxidized C. beijerinckii flavodoxin, the Gly57-Asp58 peptide adopts the O-down orientation and isomerizes to the cis conformation. This cis form is preferentially stabilized in the crystals by intermolecular hydrogen bonding to Asn137. Structures for the mutant Asn137Ala indicate that a mixture of all three conformers, mostly O-down, exists in oxidized C. beijerinckii flavodoxin in the absence of intermolecular hydrogen bonds. Redox potentials have been manipulated by substitutions that alter the conformational energies of the bend at 56M-G-D-E. The mutation Asp58Pro was constructed to study a case where energies for cis-trans conversion would be different from that of wild type. Intermolecular interactions with Asn137 are precluded in the crystal, yet Gly57-Pro58 is cis, and O-down, when the flavin is oxidized. Reduction of the flavin induces rearrangement to the trans O-up conformation. Redox potential shifts reflect the altered energies associated with the peptide rearrangement; E(ox/sq) decreases by approximately 60 mV (1.3 kcal/mol). Further, the results of mutation of Gly57 agree with predictions that a side chain at residue 57 should make addition of the first electron more difficult, by raising the energy of the O-up conformer that forms when the flavin is reduced to its semiquinone state. The ox/sq potentials in the mutants Gly57Ala, Gly57Asn, and Gly57Asp are all decreased by approximately 60 mV (1.3 kcal/mol). Introduction of the beta-branched threonine side chain at position 57 has much larger effects on the conformations and potentials. The Thr57-Asp58 peptide adopts a trans O-down conformation when the flavin is oxidized; upon reduction to the semiquinone, the 57-58 peptide rotates to a trans O-up conformation resembling that found in the wild-type protein. Changes in FMN-protein interactions and in conformational equilibria in G57T combine to decrease the redox potential for the ox/sq equilibrium by 180 mV (+4.0 kcal/mol) and to increase the sq/hq potential by 80 mV (-1.7 kcal/mol). A thermodynamic scheme is introduced as a framework for rationalizing the properties of wild-type flavodoxin and the effects of the mutations.

Structure-based perspectives on B12-dependent enzymes.

Two X-ray structures of cobalamin (B12) bound to proteins have now been determined. These structures reveal that the B12 cofactor undergoes a major conformational change on binding to the apoenzymes of methionine synthase and methylmalonyl-coenzyme A mutase: The dimethylbenzimidazole ligand to the cobalt is displaced by a histidine residue from the protein. Two methyltransferases from archaebacteria that catalyze methylation of mercaptoethanesulfonate (coenzyme M) during methanogenesis have also been shown to contain histidine-ligated cobamides. In corrinoid iron-sulfur methyltransferases from acetogenic and methanogenic organisms, benzimidazole is dissociated from cobalt, but without replacement by histidine. Thus, dimethylbenzimidazole displacement appears to be an emerging theme in cobamide-containing methyltransferases. In methionine synthase, the best studied of the methyltransferases, the histidine ligand appears to be required for competent methyl transfer between methyl-tetrahydrofolate and homocysteine but dissociates for reductive reactivation of the inactive oxidized enzyme. Replacement of dimethylbenzimidazole by histidine may allow switching between the catalytic and activation cycles. The best-characterized B12-dependent mutases that catalyze carbon skeleton rearrangement, for which methylmalonyl-coenzyme A mutase is the prototype, also bind cobalamin cofactors with histidine as the cobalt ligand, although other cobalamin-dependent mutases do not appear to utilize histidine ligation. It is intriguing to find that mutases, which catalyze homolytic rather than heterolytic cleavage of the carbon-cobalt bond, can use this structural motif. In methylmalonylCoA mutase a significant feature, which may be important in facilitating homolytic cleavage, is the long cobalt-nitrogen bond linking histidine to the co-factor. The intermediate radical species generated in catalysis are sequestered in the relatively hydrophilic core of an alpha/beta barrel domain of the mutase.

Interaction of Escherichia coli cobalamin-dependent methionine synthase and its physiological partner flavodoxin: binding of flavodoxin leads to axial ligand dissociation from the cobalamin cofactor.

Cobalamin-dependent methionine synthase from Escherichia coli catalyzes the last step in de novo methionine biosynthesis. Conversion of the inactive cob(II)alamin form of the enzyme, formed by the occasional oxidation of cob(I)alamin during turnover, to an active methylcobalamin-containing form requires a reductive methylation of the cofactor in which an electron is supplied by reduced flavodoxin and the methyl group is derived from S-adenosyl-L-methionine. E. coli flavodoxin acts specifically in this activation reaction, and neither E. coli ferredoxin nor flavodoxin from the cyanobacterium Synechococcus will substitute, despite their highly similar midpoint potentials for one-electron transfer. As assessed by EPR spectroscopy, the binding of flavodoxin to cob(II)alamin methionine synthase results in a change in the coordination geometry of the cobalt from five-coordinate to four-coordinate. Histidine 759 of methionine synthase, which replaces the normal lower ligand dimethylbenzimidazole on binding of methylcobalamin to methionine synthase, is dissociated from the cobalt of the cobalamin by the binding of flavodoxin. The association of flavodoxin and methionine synthase depends on ionic strength and pH; the pH dependence corresponds to the uptake of one proton on association. The formation of a complex between flavodoxin and methionine synthase perturbs the midpoint potentials of the flavin and cobalamin cofactors only marginally and without any significant thermodynamic advantage for electron transfer to the cobalamin of methionine synthase. No significant binding was seen between oxidized flavodoxin and methylcobalamin methionine synthase. A model for the interaction of methionine synthase with flavodoxin is proposed in which flavodoxin binding leads to changes in the distribution of methionine synthase conformations.