Lyndal M. R. Jensen

In crystallo posttranslational modification within a MauG/pre-methylamine dehydrogenase complex.

Diheme Conversion A dehydrogenase enzyme found in methylotrophic and autotrophic bacteria, which converts methylamine to ammonia and formaldehyde, must be posttranslationally modified to create a covalently bound cofactor. Synthesis of the cofactor is completed by a diheme enzyme, MauG, which oxidatively links two tryptophan residues to form tryptophan tryptophylquinone. Jensen et al. (p. 1392; see the Perspective by Bollinger and Matthews) describe the crystal structure of a catalytically competent complex of MauG bound to the methylamine dehydrogenase precursor. The reactive tryptophans are buried and well-separated from both heme irons. The heme closest to the nascent tryptophylquinone site is a 6-coordinate with an unusual His-Tyr axial link. The axial Tyr likely stabilizes a bis-Fe(IV) state involved in catalysis. Two other tyrosine residues are positioned to participate in long-range interprotein electron and radical transfer. Bacterial ammonia and formaldehyde production requires prior processing of a dehydrogenase to form a cofactor. MauG is a diheme enzyme responsible for the posttranslational modification of two tryptophan residues to form the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase (MADH). MauG converts preMADH, containing monohydroxylated βTrp57, to fully functional MADH by catalyzing the insertion of a second oxygen atom into the indole ring and covalently linking βTrp57 to βTrp108. We have solved the x-ray crystal structure of MauG complexed with preMADH to 2.1 angstroms. The c-type heme irons and the nascent TTQ site are separated by long distances over which electron transfer must occur to achieve catalysis. In addition, one of the hemes has an atypical His-Tyr axial ligation. The crystalline protein complex is catalytically competent; upon addition of hydrogen peroxide, MauG-dependent TTQ synthesis occurs.

Mutagenesis of tryptophan199 suggests that hopping is required for MauG-dependent tryptophan tryptophylquinone biosynthesis

The diheme enzyme MauG catalyzes the posttranslational modification of the precursor protein of methylamine dehydrogenase (preMADH) to complete biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Catalysis proceeds through a high valent bis-Fe(IV) redox state and requires long-range electron transfer (ET), as the distance between the modified residues of preMADH and the nearest heme iron of MauG is 19.4 Å. Trp199 of MauG resides at the MauG-preMADH interface, positioned midway between the residues that are modified and the nearest heme. W199F and W199K mutations did not affect the spectroscopic and redox properties of MauG, or its ability to stabilize the bis-Fe(IV) state. Crystal structures of complexes of W199F/K MauG with preMADH showed no significant perturbation of the MauG-preMADH structure or protein interface. However, neither MauG variant was able to synthesize TTQ from preMADH. In contrast, an ET reaction from diferrous MauG to quinone MADH, which does not require the bis-Fe(IV) intermediate, was minimally affected by the W199F/K mutations. W199F/K MauGs were able to oxidize quinol MADH to form TTQ, the putative final two-electron oxidation of the biosynthetic process, but with kcat/Km values approximately 10% that of wild-type MauG. The differential effects of the W199F/K mutations on these three different reactions are explained by a critical role for Trp199 in mediating multistep hopping from preMADH to bis-Fe(IV) MauG during the long-range ET that is required for TTQ biosynthesis.

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Despite the importance of tryptophan (Trp) radicals in biology, very few radicals have been trapped and characterized in a physiologically meaningful context. Here we demonstrate that the diheme enzyme MauG uses Trp radical chemistry to catalyze formation of a Trp-derived tryptophan tryptophylquinone cofactor on its substrate protein, premethylamine dehydrogenase. The unusual six-electron oxidation that results in tryptophan tryptophylquinone formation occurs in three discrete two-electron catalytic steps. Here the exact order of these oxidation steps in the processive six-electron biosynthetic reaction is determined, and reaction intermediates are structurally characterized. The intermediates observed in crystal structures are also verified in solution using mass spectrometry. Furthermore, an unprecedented Trp-derived diradical species on premethylamine dehydrogenase, which is an intermediate in the first two-electron step, is characterized using high-frequency and -field electron paramagnetic resonance spectroscopy and UV-visible absorbance spectroscopy. This work defines a unique mechanism for radical-mediated catalysis of a protein substrate, and has broad implications in the areas of applied biocatalysis and understanding of oxidative protein modification during oxidative stress.

Functional importance of tyrosine 294 and the catalytic selectivity for the bis-Fe(IV) state of MauG revealed by replacement of this axial heme ligand with histidine .

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high-valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His, and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in interprotein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation affects not only the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 Å separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high-valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)═O/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)═O heme but via long-range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high-valent heme iron.

Proline 107 is a major determinant in maintaining the structure of the distal pocket and reactivity of the high-spin heme of MauG.

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies had shown that Pro107, which resides in the distal pocket of the high-spin heme of MauG, changes conformation upon binding of CO or NO to the heme iron. In this study, Pro107 was converted to Cys, Val, and Ser by site-directed mutagenesis. The structures of each of these MauG mutant proteins in complex with preMADH were determined, as were their physical and catalytic properties. P107C MauG was inactive, and the crystal structure revealed that Cys107 had been oxidatively modified to a sulfinic acid. Mass spectrometry revealed that this modification was present prior to crystallization. P107V MauG exhibited spectroscopic and catalytic properties that were similar to those of wild-type MauG, but P107V MauG was more susceptible to oxidative damage. The P107S mutation caused a structural change that resulted in the five-coordinate high-spin heme being converted to a six-coordinate heme with a distal axial ligand provided by Glu113. EPR and resonance Raman spectroscopy revealed this heme remained high-spin but with greatly increased rhombicity as compared to that of the axial signal of wild-type MauG. P107S MauG was resistant to reduction by dithionite and reaction with H(2)O(2) and unable to catalyze TTQ biosynthesis. These results show that the presence of Pro107 is critical in maintaining the proper structure of the distal heme pocket of the high-spin heme of MauG, allowing exogenous ligands to bind and directing the reactivity of the heme-activated oxygen during catalysis, thus minimizing the oxidation of other residues of MauG.

Structures of MauG in complex with quinol and quinone MADH

MauG has been cocrystallized with methylamine dehydrogenase (MADH) with its TTQ cofactor in the o-quinol (TTQOQ) and quinone (TTQOX) forms and the structures of the resulting complexes have been solved. The TTQOQ structure crystallized in either space group P21 or C2, while the TTQOX structure crystallized in space group P1. The previously solved structure of MauG in complex with MADH bearing an incompletely formed TTQ cofactor (preMADH) also crystallized in space group P1, although with different unit-cell parameters. Despite the changes in crystal form, the structures are virtually identical, with only very minor changes at the protein-protein interface. The relevance of these structures with respect to the measured changes in affinity between MauG and various forms of MADH is discussed.

A Trp199Glu MauG variant reveals a role for Trp199 interactions with pre-methylamine dehydrogenase during tryptophan tryptophylquinone biosynthesis

MauG catalyzes posttranslational modifications of a methylamine dehydrogenase precursor (preMADH) to complete the biosynthesis of its protein‐derived tryptophan tryptophylquinone (TTQ) cofactor. Trp199 is present at the site of interaction between MauG and preMADH and is critical to this process as it mediates hole hopping during the inter‐protein electron transfer that is required for catalysis. Trp199 was converted to Glu and the structure and reactivity of the W199E/preMADH complex were characterized. The results reveal that the nature of residue 199 is also important for productive complex formation between preMADH and MauG.

MauG catalysis: a tale of ferryl iron, radicals and long distance hopping

Methylamine dehydrogenase (MADH) enables some methylotrophic/autotrophic bacteria to grow on methylamine as a sole source of carbon and energy. MADH catalysis depends on the cofactor tryptophan tryptophylquinone (TTQ) that is a posttranslational modification of two Trp residues in the MADH β-subunit. The maturation of MADH depends on four gene products located in the methylamine utilization (mau) gene cluster. One of these, mauG, encodes a c-type di-heme enzyme that completes synthesis of the TTQ cofactor. The potent oxidant is an unusual bis-Fe(IV) MauG species composed of a ferryl heme (Fe(IV)=O) with an oxidizing equivalent stored as Fe(IV) at the second heme, which has an unusual His, Tyr axial ligation. The bis-Fe(IV) oxidant is formally Fe(V) and equivalent to Compound I. Completion of TTQ to generate active MADH involves long-range electron transfer and a radical hopping mechanism to effect catalysis over a 40 Å distance. The MauG catalyzed reaction occurs in three discrete 2-electron events in a hydrogen peroxide or molecular oxygen (+ reducing equivalents) dependent process. A crystal structure of MauG in complex with its protein substrate, a precursor form of MADH known as preMADH, has been solved. The crystals are catalytically active. The order of the 2-electron chemistry catalyzed by MauG was determined through a series of structures from crystals harvested after different amounts of time following crystallization. Hydrogen peroxide to initiate the reaction was generated by the slow breakdown of polyethylene glycol used in crystallization. These in crystallo data are corroborated by mass spectrometry in solution experiments.