In cell extracts of Micrococcus flavus and Arthrobacter sp. KM, inorganic polyphosphate [poly (P)]- and ATP-dependent NADH kinase activities were detected, and both activities were confirmed to be catalyzed by poly (P)/ATP-NAD kinase, but not by NADH-specific kinase. Poly (P)/ATP-NAD kinas from Mycobacterium tuberculosis also exhibited NADH kinase activity. On the other hand, ATP-NAD kinase from Saccharomyces cerevisiae catalyzed the NADH kinase reaction, although that of Escherichia coli showed no NADH kinase activity, nor did a cell extract of E. coli. Thus, it is indicated that poly (P)/ATP-NAD kinase possesses NADH kinase activity in addition to NAD kinase one, while the ability of ATP-NAD kinase to catalyze the NADH kinase reaction differs in microbes that contain the enzyme.

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Over the recent decade, a number of unique cofactors derived from amino acid residues have been discovered in various enzyme proteins, including 2,4,5-trihydroxyphenylalanine quinone (topa quinone; TPQ) of copper amine oxidase and tryptophan tryptophylquinone (TTQ) of bacterial amine dehydrogenase. These quinone cofactors temporarily store the reducing equivalents derived from the substrate before passing them on to exogenous 1- or 2-electron acceptors such as a cytochrome, a cupredoxin, or molecular oxygen. Both of TPQ and TTQ are encoded as normal amino acids (Tyr or Trp) in the enzyme genes and thus are synthesized by post-translational modification. Using the inactive precursor forms of recombinant copper amine oxidases from Arthrobacter globiformis overproduced in Escherichia coli, we previously demonstrated that TPQ is generated through self-processing of the enzyme proteins with the participation of the bound copper ion. X-ray crystallographic studies of the Cu/TPQ-less inactive enzyme (apoenzyme) as well as the Cu/TPQ-containing active form (holoenzyme) revealed the structural difference only in the active site. Furthermore, we have recently identified a novel quinone cofactor, cysteine tryptophylquinone (CTQ) in quinohemoprotein amine dehydrogenases from Paracoccus denitrificans and Pseudomonas putida. CTQ is the forth quinone cofactor derived from amino acids, following TPQ, TTQ, and lysine tyrosylquinone (LTQ) found in lysyl oxidase.

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Cysteine has been shown to be the source of sulfur for the biosyntheses of a variety of cofactors including biotin, lipoic acid, molybdopterin, and thiamine, as well as Fe-S clusters in proteins and thionucleosides in tRNA. Although the source has been identified, the biochemical steps for sulfur incorporation into these molecules are poorly understood. However, recent studies have provided evidence that a pyridoxal 5^1-phosphate-dependent cysteine desulfurase is involved in the initial stages of sulfur trafficking within cells. Cysteine desulfurase is a homodimeric enzyme which catalyzes the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. The persulfide intermediate donates sulfur to Fe-S clusters, thiamine, and thionucleosides in tRNA. The enzyme is also proposed to be involved in cellular iron homeostasis and in the biosynthesis of selenoproteins. This paper describes recent developments in the study of cysteine desulfurases.

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Certain enzymes utilize the high reactivity of radicals to catalyze chemically difficult reactions. Coenzyme B_<12> serves as a cofactor for enzymatic radical reactions. The three-dimensional structures of coenzyme B_<12>-dependent diol dehydratase and glycerol dehydratase were determined by X-ray crystallography. The structure-based fine mechanism of action of diol dehydratase was studied to establish the general mechanism for B_<12> enzymes as well as radical enzymes. The steric strain model was proposed for the coenzyme cobalt-carbon bond homolysis. The ribosyl rotation model well explained the distance problem and the stereospecificity in hydrogen abstraction. The substrate-induced conformational change of the enzyme revealed the substrate triggering mechanism for the catalytic radical formation. Theoretical calculations as well as mutational studies suggested that the hydroxyl group migrates by the concerted pathway through a three-membered cyclic transition state which is stabilized by active-site amino acid residues. A refined catalytic mechanism for diol dehydratase is proposed here.

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On the bases of the crystal structures of flavoenzymes, D-amino acid oxidase (DAO) and acyl-CoA oxidase (AGO), which have recently been solved by us, the control mechanisms of the flavin reactivity toward molecular oxygen in the oxidative half-reaction are discussed. The three-dimensional structure of the purple intermediate of DAO, the requisite intermediate of the oxidative half-reaction, reveals the electrostatic effect on raising the electron density at C (4a) of the anionic reduced flavin. The high electron density at C (4a), the site of electron donation to oxygen, results in enhanced reactivity of reduced flavin toward molecular oxygen. According to the three-dimensional structure of rat liver AGO, the active site cleft is larger than that of acyl-CoA dehydrogenase allowing easy access of molecular oxygen in the oxidative half-reaction and formation of the electron-transfer complex with an electron acceptor protein is effectively blocked, rendering AGO the oxidase rather than the dehydrogenase functionality.

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