William S. McIntire

What's in a covalent bond? On the role and formation of covalently bound flavin cofactors

Many enzymes use one or more cofactors, such as biotin, heme, or flavin. These cofactors may be bound to the enzyme in a noncovalent or covalent manner. Although most flavoproteins contain a noncovalently bound flavin cofactor (FMN or FAD), a large number have these cofactors covalently linked to the polypeptide chain. Most covalent flavin–protein linkages involve a single cofactor attachment via a histidyl, tyrosyl, cysteinyl or threonyl linkage. However, some flavoproteins contain a flavin that is tethered to two amino acids. In the last decade, many studies have focused on elucidating the mechanism(s) of covalent flavin incorporation (flavinylation) and the possible role(s) of covalent protein–flavin bonds. These endeavors have revealed that covalent flavinylation is a post‐translational and self‐catalytic process. This review presents an overview of the known types of covalent flavin bonds and the proposed mechanisms and roles of covalent flavinylation.

[37] Covalent attachment of flavin to flavoproteins: Occurrence, assay, and synthesis☆

Publisher Summary This chapter describes occurrence, assay, and synthesis of covalent attachment of flavin to flavoproteins. The first covalently bound flavin to be discovered was the flavin component of mammalian succinate dehydrogenase. Five different structures have been identified in this group of enzymes and chemically synthesized; four are adducts formed with the 8 α -methyl group of the flavin ring system, and the fifth is a substituent at C(6) of the aromatic ring of the flavin. It is essential to purify the flavin peptide first to remove materials which interfere in subsequent tests, such as hemes originating from cytochromes. To eliminate losses by having the flavin present in several phosphorylated forms, as a result of breakdown during manipulation, it is desirable to convert the crude peptide to the dephosphorylated form, by sequential treatment with nucleotide pyrophosphatase and alkaline phosphatase. Advantage may be taken of the different R f values of flavin peptides in these systems for the monophosphorylated and diphosphorylated forms, so that the procedure may be carried out both before and after digestion with the phosphatases. A more sensitive procedure for histidylflavins is based on the difference in fluorescence of the aminoacyl flavin between pH 3.2 and 7.

Organization and sequences of p-hydroxybenzaldehyde dehydrogenase and other plasmid-encoded genes for early enzymes of the p-cresol degradative pathway in Pseudomonas putida NCIMB 9866 and 9869.

The gene (designated pchA) encoding the aldehyde dehydrogenase that is required to metabolise the p-hydroxybenzaldehyde produced by the degradation of p-cresol in Pseudomonas putida NCIMB 9866 and 9869 has been identified on plasmids pRA4000 and pRA500, respectively. The gene lies immediately upstream of the pchC and pchF genes encoding the subunits of p-cresol methylhydroxylase (PCMH), the preceeding enzyme in the p-cresol degradative pathway. In pRA500 the latter genes are followed by the genes encoding the alpha (pcaG) and beta (pcaH) subunits of protocatechuate-3,4-dioxygenase, whereas in pRA4000 the genes encoding PCMH are followed by an open reading frame encoding a protein that is similar to the maturase-related protein of P. alcaligenes. A gene, designated pchX, that encodes a protein of unknown function was identified between the pchC and pchF genes in both plasmids.

cDNA Sequences of Variant Forms of Human Placenta Diamine Oxidase

Genes for two forms of human placenta diamine oxidase(dao) were cloned from a cDNA library and sequenced. One gene,pdao 1, is identical in length to human kidneydao but differs from it by two bases in the coding region and differs slightly in the 3′- and 5′-noncoding regions. The second gene,pdao2, is nearly identical to these genes in the coding region, except that it has an extra 57-nucleotide coding segment near the 3′ end of this region. This segment corresponds to the contiguous sequence of the 3′ end of intron 3 of human kidneydao. pdao2 also differs significantly frompdao1 and human kidneydao in a 13-base sequence in the 5′-noncoding region. It is proposed thatpdao1 and human kidneydao are polymorphic forms of the same allele. Whetherpdao2 is a polymorph of these two is not certain, because of the significant differences in the coding and noncoding regions.Pdao2 may represent a different allele.

AMINE-OXIDIZING QUINOPROTEINS

Publisher Summary This chapter describes the five types of amine-oxidizing quinoproteins—(1) lupanine 17-hydroxylase, which contains pyrroloquinoline quinone (PQQ) proteins; (2) alkylamine dehydrogenases, which contain tryptophan tryptophylquinone (TTQ) proteins; (3) copper-containing alkylamine and diamine oxidases, which contain topa quinone (TPQ) proteins; (4) semicarbazide-sensitive amine oxidases; and (5) lysyl oxidase, which contains lysine tyrosylquinone (LTQ). Lupanine 17-hydroxylase, from Pseudomonas lupanini, is the only amine oxidizer to use PQQ as its redox cofactor. All other PQQuinoproteins are aldehyde or alcohol dehydrogenases. All TTQuinoproteins are alkylamine dehydrogenases. Primary alkylamines are oxidized to the corresponding aldehydes, with concomitant reduction of TTQ. The reduced quinone transfers electrons to a reoxidizing substrate. Several new TPQuinoproteins have been isolated, and many more have been cloned and/or their genes heterologously expressed. These include gene cloning and expression, and characterization of human kidney diamine oxidase (amiloride-binding protein); gene cloning and sequencing of two forms of human placenta diamine oxidase, one of which is essentially identical to human kidney diamine oxidase and; cloning of a human placental monoamine oxidase gene, which maps to chromosome 17q21.