The Covalent Structure of the Small Subunit fromPseudomonas putida Amine Dehydrogenase Reveals the Presence of Three Novel Types of Internal Cross-linkages, All Involving Cysteine in a Thioether Bond

Abstract

Pseudomonas putida contains an amine dehydrogenase that is called a quinohemoprotein as it contains a quinone and two hemes c as redox active groups. Amino acid sequence analysis of the smallest (8.5 kDa), quinone-cofactor-bearing subunit of this heterotrimeric enzyme encountered difficulties in the interpretation of the results at several sites of the polypeptide chain. As this suggested posttranslational modifications of the subunit, the structural genes for this enzyme were determined and mass spectrometric de novo sequencing was applied to several peptides obtained by chemical or enzymatic cleavage. In agreement with the interpretation of the X-ray electronic densities in the diffraction data for the holoenzyme, our results show that the polypeptide of the small subunit contains four intrachain cross-linkages in which the sulfur atom of a cysteine residue is involved. Two of these cross-linkages occur with the ␤-carbon atom of an aspartic acid, one with the ␥-carbon atom of a glutamic acid and the fourth with a tryptophanquinone residue, this adduct constituting the enzyme's quinone cofactor, CTQ. The thioether type bond in all four of these adducts has never been found in other proteins. CTQ is a novel cofactor in the series of the recently discovered quinone cofactors.A diversity of enzymes appears to be involved in amine oxidation, as reflected by the number of different cofactors found in the types established so far (1). Based on the natural electron acceptor used, a further distinction can be made between amine oxidases and amine dehydrogenases. Both classes convert amines into their corresponding aldehydes, but oxidases produce toxic peroxides, while the reducing equivalents in the case of dehydrogenases are directly transferred to the respiratory chain (2).Depending on the identity of their cofactor(s), amine dehydrogenases are subdivided into quinoproteins, flavoproteins, quinohemoproteins, and flavohemoproteins (2). Pseudomonas putida strain ATCC 12633, as well as strain IFO 15633, contain a novel type of amine dehydrogenase; a quinohemoprotein (QH-AmDH) 1 as a quinone compound is present in the small subunit and two heme c groups in the large subunit (3). Although the quinone cofactor was not liberated on denaturing the enzyme, spectroscopic data (4) already indicated that it is different from tryptophan tryptophylquinone (5), topaquinone (6), or lysine tyrosylquinone (7), forming part of the protein chain of several amine dehydrogenases (EC 1.4.99.3/4), several amine oxidases (EC 1.4.3.6), and protein-lysine 6-oxidase (EC 1.4.3.13), respectively.To reveal the identity of the quinone cofactor and its position in the protein, the genes for QH-AmDH were cloned and sequenced, and the small subunit subjected to chemical analysis. The latter was carried out in a combination of automated Edman degradation, mass spectrometry, liquid chromatography, and fragmentation MS applied to the underivatized and the derivatized form (the quinone cofactor converted into a hydrazone with a hydrazine) of the small sub...

Crystal Structure of Quinohemoprotein Amine Dehydrogenase from Pseudomonas putida

“…2C). Crystallographic identification of these cross-linked structures greatly enabled us to interpret the results obtained by the chemical and mass spectrometric analyses of the ␥-subunit, as reported recently (10). Besides the novel CTQ cofactor encaged by multiple internal cross-bridges, another intriguing feature is that the ␥-subunit scarcely contains regular secondary structures as it has only two short ␣-helices with many turns and bends (Fig.…”

“…As deduced from the DNA sequence (10), this 79-residue subunit contains a total of four Cys and five Trp residues. However, neither free SH groups nor S-S bridges were detected in it by chemical analysis (10). Furthermore, during the initial stage of the structure determination by x-ray crystallography, modeling of the ␥-subunit puzzled us with its main chain branching off at many points in the electron density map.…”

“…Overall Structure-The crystal structure of the native QHAmDH was determined at 1.9-Å resolution (Tables I and II), utilizing the DNA-based protein sequence (10). Consistent with the previous biochemical analysis (7), the enzyme is composed of three non-identical subunits: a 494-residue ␣-subunit (ϳ60 kDa) carrying two heme c groups, a 349-residue ␤-subunit (ϳ40 kDa), and a 79-residue ␥-subunit (ϳ9 kDa) bearing the quinone cofactor.…”

“…To identify the quinone cofactor and its position in the protein, we (10) have recently determined the primary structure of the quinone-containing small subunit of QH-AmDH from P. putida using a combination of automated Edman degradation and mass spectrometry, and we have also cloned the genes coding for the three subunits of the heterotrimeric enzyme. Although we initially encountered difficulties in the interpretation of the chemical and mass data, the progress in elucidating the crystal structure of the enzyme enabled us to unequivocally identify a novel quinone cofactor, cysteine tryptophylquinone (CTQ) (naming is analogous with that for TTQ).…”

“De novo” peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: A preliminary study

“…Notable in this regard is the use of 18 O incorporation at the carboxyl-termini of peptides during protein hydrolysis [14 -17]. For underivatized peptides, ESI has found use for de novo sequencing under a variety of conditions, which include collision-induced dissociation (CID) in the triple quadrupole mass analyzer [18 -20] and the QqTOF mass analyzer [18,19,[21][22][23][24][25][26], resonant excitation in the quadrupole ion trap mass analyzer [14,[27][28][29], and electron capture dissociation in the Fourier transform ion cyclotron resonance mass analyzer [30 -34]. MALDI de novo sequencing has been carried out using CID and metastable decay in single- [35] and double-stage [36 -39] TOF instruments and with CID in QqTOF instruments [40 -44].…”