Flavoenzymes: diverse catalysts with recurrent features

Fraaije, Marco W.; Mattevi, Andrea

doi:10.1016/s0968-0004(99)01533-9

Cited by 473 publications

(523 citation statements)

References 39 publications

Supporting

Mentioning

491

Contrasting

Unclassified

Order By: Relevance

“…The C1 atom of the lactam moiety, which corresponds to the site of oxidative attack in cellobiose, binds in a position 2.9 Å in front of and below the N5-C4a locus of the isoalloxazine ring, defining an angle of 108°(molecule A) and 110°(molecule B) with the N5-N10 atoms of the flavin ring. These values are in agreement with those typically observed in flavoenzymes (30). It should be noted that the lactam C1 and O1 are almost perfectly aligned with the flavin N5 and C4a, respectively (C1-N5, 2.9 Å; O1-C4a, 2.9 Å).…”

Section: Resultssupporting

confidence: 79%

Mechanism of the Reductive Half-reaction in Cellobiose Dehydrogenase

Hällberg¹,

Henriksson²,

Pettersson³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The extracellular flavocytochrome cellobiose dehydrogenase (CDH; EC 1.1.99.18) participates in lignocellulose degradation by white-rot fungi with a proposed role in the early events of wood degradation. The complete hemoflavoenzyme consists of a catalytically active dehydrogenase fragment (DH cdh ) connected to a b-type cytochrome domain via a linker peptide. In the reductive half-reaction, DH cdh catalyzes the oxidation of cellobiose to yield cellobiono-1,5-lactone. The active site of DH cdh is structurally similar to that of glucose oxidase and cholesterol oxidase, with a conserved histidine residue positioned at the re face of the flavin ring close to the N5 atom. The mechanisms of oxidation in glucose oxidase and cholesterol oxidase are still poorly understood, partly because of lack of experimental structure data or difficulties in interpreting existing data for enzyme-ligand complexes. Here we report the crystal structure of the Phanerochaete chrysosporium DH cdh with a bound inhibitor, cellobiono-1,5-lactam, at 1.8-Å resolution. The distance between the lactam C1 and the flavin N5 is only 2.9 Å, implying that in an approximately planar transition state, the maximum distance for the axial 1-hydrogen to travel for covalent addition to N5 is 0.8 -0.9 Å. The lactam O1 interacts intimately with the side chains of His-689 and Asn-732. Our data lend substantial structural support to a reaction mechanism where His-689 acts as a general base by abstracting the O1 hydroxyl proton in concert with transfer of the C1 hydrogen as hydride to the re face of the flavin N5.Cellobiose dehydrogenases (CDHs 1 ; EC 1.1.99.18) are extracellular fungal flavocytochromes that are believed to participate in lignocellulose degradation by fungi. They are oxidoreductases carrying protoheme and FAD cofactors bound to separate domains. In vitro, CDH from the white-rot Basidiomycete Phanerochaete chrysosporium depolymerizes cellulose, hemicelluloses, and lignin (Refs. 1-3; for review, see Ref. 4) as well as other polymers (5). The exact biological function of CDH has been a subject of lively debate, but recent results suggest that the enzyme is important for invasion and colonization of wood (6).The catalytic site is located in the flavoprotein domain, where the reductive half-reaction proceeds by oxidation of ␤-cellobiose (apparent k cat 15.7 s Ϫ1 and K m 0.11 mM, see Ref. 7) to yield cellobiono-1,5-lactone (Fig.

show abstract

Section: Resultssupporting

confidence: 79%

Mechanism of the Reductive Half-reaction in Cellobiose Dehydrogenase

Hällberg¹,

Henriksson²,

Pettersson³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Disruption of the electrostatic interaction between the amino acid carboxylate and Arg98 would be expected to result in less than optimal positioning of the substrate in the active site. In a number of flavoproteins which catalyze transfer of a hydride equivalent to the flavin, the carbon from which the hydrogen is lost binds an average of 3.5 Å from flavin N5 (20), consistent with a requirement for a precise alignment of the amino acid and flavin in the active site. Loss of the arginine residue responsible for anchoring the carboxylate of the amino acid would disrupt this positioning, with a consequent decrease in the rate of catalysis.…”

Section: Discussionmentioning

confidence: 85%

Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family

Sobrado

Fitzpatrick

2003

Biochemistry

View full text Add to dashboard Cite

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. We have previously identified tryptophan 2-monooxygenase as a homologue of L-amino acid oxidase [Sobrado, P., and Fitzpatrick, P. F. (2002) Arch. Biochem. Biophys. 402,[24][25][26][27][28][29][30]. On the basis of the sequence comparisons of the different LAAO family members, Arg98 of tryptophan 2-monooxygenase can be identified as an active site residue which interacts with the carboxylate of the amino acid substrate. The catalytic properties of R98K and R98A tryptophan 2-monooxygenase have been characterized to evaluate the role of this residue. Mutation of Arg98 to lysine decreases the first-order rate constant for flavin reduction by 180-fold and the second-order rate constant for flavin oxidation by 26-fold, has no significant effect on the K d value for tryptophan or the K i value for the competitive inhibitor indoleacetamide, and increases the K i value for indolepyruvate less than 2-fold. Mutation of this residue to alanine decreases the rate constants for reduction and oxidation an additional 5-and 2-fold, respectively, and increases the K d value for tryptophan and the K i value for indolepyruvate by 31-and 17-fold, respectively, while having an only 2-fold effect on the K i value for indoleacetamide. Both mutations increase the value of the primary deuterium isotope effect with tryptophan as a substrate, consistent with a later transition state. Both mutant enzymes catalyze a simple oxidase reaction, producing indolepyruvate and hydrogen peroxide. The pH dependences of the V/K trp values for the mutant enzymes show that the anionic form of the substrate is preferred but that the zwitterionic form is a substrate. The results are consistent with the interaction between Arg98 and the carboxylate of the amino acid substrate being critical for correct positioning of the substrate in the active site for efficient catalysis.Tryptophan 2-monooxygenase (TMO) 1 is an FAD-containing enzyme that catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide (Scheme 1). This reaction is the first step in the biosynthesis of the plant hormone indoleacetic acid by plant pathogens such as Agrobacterium tumefaciens and Pseudomonas savastanoi; the resulting production of indoleacetic acid induces the formation of galls at the site of infection (1,2). The kinetic mechanism of TMO has been determined with tryptophan as a substrate and can be divided † This work was supported in part by grants to P.F.F. from the NIH (GM 58698) and from the Robert A. Welch Foundation (A-1245) and by a fellowship from CONICIT-Costa Rica (02-2002) to P.S. * To whom correspondence should be addressed: Department of Biochemistry and Biophysics, 2128 TAMU, College Station, TX 77843-2128. Phone: (979) into two half-reactions (Scheme 2). In the reductive half-reaction, the α-C-H bond of the amino acid is broken and a hydride equivalent is transferred to the FAD, forming the reduced enzyme with the imin...

show abstract

“…ring in the high-resolution mMICAL 489 structure is in the out position (24). In contrast, the position of the flavin ring in most MO structures corresponds to the in conformation of PHBH (10), which places the reactive isoalloxazine in position to contribute to catalysis. Some MOs are permanently locked into the in conformation, but, for the PHBH family of hydroxylases, the ability to switch between in and out conformations is essential to allow access to the active site for substrate binding and product release.…”

Section: Resultsmentioning

confidence: 99%

“…Despite Ͻ20% sequence identity between disparate members of this family, they share a similar fold and essentially identical FAD-binding sites (10). In contrast, the catalytic reactions carried out by the flavoenzymes are varied, and their active-site architectures differ accordingly.…”

mentioning

confidence: 99%

High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule

Siebold

Berrow

Walter

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

123

View full text Add to dashboard Cite

MICALs are large (Ͼ1,000 aa), multidomain, cytosolic proteins expressed in specific neuronal and nonneuronal (thymus, lung, spleen, and testis) tissues both during development and in adulthood (4).From sequence analysis, it has been shown that MICALs contain two protein-protein interaction domains implicated in signal transduction and cytoskeletal organization, a calponin homology (CH) domain (7) and a LIM domain (8), plus a proline-rich region for Src homology 3 (SH3) domain recognition that mediates interaction with CasL, a multidomain docking protein localized at focal adhesions and stress fibers (4). Human MICAL-1 associates with the small GTPase Rab1 (6, 9) and with vimentin (4), a major component of intermediate filaments. In addition to the SH3 domainbinding motif, the C-terminal region (of Ϸ250 residues) contains coiled-coil motifs and binds the cytosolic domain of class A plexins (5). Thus, the MICALs are protein-binding scaffolds, but, uniquely, they combine this property with a highly conserved N-terminal region of some 500 residues, characterized by sequence analyses and functional studies as a putative flavoprotein monooxygenase (MO) required for semaphorin-plexin-mediated axon guidance (5).Flavoenzymes bind the cofactor FAD as an integral part of their structure. Despite Ͻ20% sequence identity between disparate members of this family, they share a similar fold and essentially identical FAD-binding sites (10). In contrast, the catalytic reactions carried out by the flavoenzymes are varied, and their active-site architectures differ accordingly. The structure of p-hydroxybenzoate hydroxylase (PHBH) provides the paradigm for the flavoprotein MO (hydroxylase) subset of flavoenzymes (11). Flavoprotein MOs act on a broad range of small molecules (e.g., phydroxybenzoate, steroids, and amino acids). The substrate(s), mode of action, and, indeed, function of the putative MO region in the MICALs are unknown.Our structural and biophysical analyses on the N-terminal portion of murine MICAL-1 confirm that this region has the architecture and characteristics of a flavoenzyme of the MO family, demonstrate the enzymatic activity to be NADPH-dependent, and reveal a mechanism for controlled substrate access to the active site, which is strongly indicative of large (potentially protein) substrates. MethodsProtein Expression and Purification. The mMICAL 489 expression construct (amino acids 1-489 of the mouse MICAL-1 gene plus C-terminal His-tag) was generated by ligation-independent cloning (Gateway Technology, Invitrogen), overexpressed in Escherichia coli (DE3)pLysS (Novagen), and purified with Ni affinity and size-exclusion chromatography; all stages used the high-throughput pipeline of the Oxford Protein Production Facility (see Supporting Text, which is published as supporting information on the PNAS

show abstract

Flavoenzymes: diverse catalysts with recurrent features

Cited by 473 publications

References 39 publications

Mechanism of the Reductive Half-reaction in Cellobiose Dehydrogenase

Mechanism of the Reductive Half-reaction in Cellobiose Dehydrogenase

Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family

High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule

Contact Info

Product

Resources

About