ADP Competes with FAD Binding in Putrescine Oxidase

Abstract

Putrescine oxidase (PuO, 4 EC 1.4.3.10) is a flavin-containing amine oxidase involved in polyamine degradation. It catalyzes the oxidative deamination of putrescine (1,4-diaminobutane) into 4-aminobutanal with concomitant reduction of oxygen to hydrogen peroxide (Scheme 1). In addition, the oxidase also accepts other polyamines, like spermine and spermidine, which are involved in cell growth and differentiation and interact with nucleic acids (1, 2).PuO was first reported in the 1960s when it was isolated from Micrococcus rubens (3, 4). This enzyme (PuO Mr ) is a soluble homodimeric protein and is unique among flavoprotein oxidases in that the isolated enzyme appears to contain only one noncovalently bound FAD molecule per dimeric protein (5). PuO Mr was heterologously expressed in Escherichia coli (6) and is applied as a diagnostic enzyme for the detection of di-and polyamines (7,8). Increased levels of polyamines in tissue and body fluids are related to cancer and thus can be used as tumor markers (9). PuO Mr can also be used to monitor food freshness by detecting polyamines (10).Recently we identified a novel putrescine oxidase from the actinomycete Rhodococcus erythropolis (PuO Rh ) (11). PuO Rh shares 67% sequence identity with PuO Mr and displays similar catalytic properties: it is highly specific for putrescine and is effectively inhibited by aliphatic monoamines and short diamines. Like PuO Mr , PuO Rh is a homodimer of about 100 kDa and, interestingly, also contains only one mol of noncovalently bound FAD per mol of dimeric protein. Such a low flavin cofactor occupancy is remarkable as inspection of the PuO sequences suggests that each monomer contains a Rossmanfold domain capable of FAD binding.To explain the reason of the unusually low FAD/protein ratio in PuO, more detailed structural information would be highly informative. Unfortunately, no crystal structure is available for a PuO. However, for the sequence-related human monoamine oxidase B (MAO-B), the crystal structure has been solved (12). MAO-B is a flavoprotein oxidase located at the outer mitochondrial membrane (13) and is involved in the oxidation of neurotransmitters (14). The human oxidase shares 32% sequence identity with PuO Rh and catalyzes the oxidative deamination of aromatic monoamines like phenylethylamine and benzylamine. Based on the MAO-B structure we have constructed a structural model for PuO Rh (11). By inspection of the model and subsequent site-directed mutagenesis studies, we have verified that one specific active site residue (Glu-324) in PuO Rh is crucial for its preference for aliphatic diamine substrates. Like PuO Mr and PuO Rh , MAO-B is a homodimeric protein, but in contrast to both bacterial oxidases, each MAO-B subunit contains one covalently bound FAD cofactor. The FAD in MAO-B is covalently attached to a cysteine residue (Cys-397) via a C8␣-linkage.In this report, we describe our findings concerning a more detailed analysis of cofactor binding in PuO Rh . The data reveal that ADP is competing with FAD binding. Fur...

Flavin–Protein Complexes: Aromatic Stacking Assisted by a Hydrogen Bond

“…Recently, it was shown that ADP competes with FAD in putrescine oxidase, and the enzyme was isolated with ADP bound at the ADP binding site of FAD. 47 If ADP is added at a sufficiently high concentration to the reconstituted apoprotein−FAD complex, the rate of ADP incorporation should be limited by the FAD dissociation rate. We used this approach to determine the FAD ox dissociation rate constants.…”

“…44−46 The apoprotein displaces the equilibrium toward the unstacked form of FAD by anchoring the 5′-ADP moiety throughout interaction with several amino acids of the consensus sequence located in the positively charged groove of the FAD binding site. The use of the ADP moiety as the major anchoring point to the apoprotein has been observed in several flavoenzymes, 47,54,55 and this phenomenon could be a general feature among the FAD binding proteins. Anchoring the ADP likely restricts the conformational sampling of the cofactor and may help the isoalloxazine to translocate into the active site.…”

What’s in a covalent bond?

“…To probe whether PuO could be converted to a covalent flavoprotein, an alanine residue corresponding to the linking cysteine in human MAO B was replaced by a cysteine. Intriguingly, the A394C PuO mutant was indeed able to form a covalent FAD–protein bond [73]. The ability to convert a noncovalent flavoprotein PuO into a covalent flavoprotein by a single amino acid replacement also confirms the self‐catalytic nature of covalent flavinylation.…”

“…Results from a recent study on a sequence‐related amine oxidase have hinted at another rationale for covalent flavinylation. The bacterial putrescine oxidase (PuO) was shown to contain equal amounts of tightly but noncovalently bound FAD and ADP [73]. On the basis of the high degree of sequence identity between mammalian MAO and PuO, only one dinucleotide cofactor is expected to bind to PuO.…”

Synthesis ofN-heterocycles from diaminesviaH2-driven NADPH recycling in the presence of O2

“…19,21 The previous low FAD loading is related to competing binding of the inhibitor adenosine diphosphate to the active site. 22 The nearly total FAD occupancy of purified PuO derivatives can be explained by optimized PuO production conditions at low temperature (18°C) in TB complex media. The UV/Vis spectra showed typical characteristics of flavoenzymes ( Fig.…”