Andrei Y. Chistoserdov

A new cofactor in a prokaryotic enzyme : Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

Methylamine dehydrogenase (MADH), an alpha 2 beta 2 enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small beta subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by the Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting 1H and 13C nuclear magnetic resonance, and mass spectral data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, or pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.

The genetic organization of the mau gene cluster of the facultative autotroph Paracoccus denitrificans

The mau gene cluster from Paracoccus denitrificans was cloned. The regions of a cloned fragment carrying genes for the small and the large subunit of the methylamine dehydrogenase were identified and sequenced. Open reading frames for the MADH small subunit gene and the MADH large subunit gene were identified. Three other open reading frames coding polypeptides with unknown function were found in the sequence. The small subunit gene sequence data reveal that the MADH small subunit polypeptide from P. denitrificans has an unusual leader sequence and contains the tryptophan tryptophyl quinone cofactor. The MADH small subunit genes and the parts of the open reading frames found upstream of them in the genome of M. extorquens AM1 and P. denitrificans have considerable similarity. The sequence data have been used for refinement of the X-ray crystallographic structure of the MADH from P. denitrificans, and key conserved residues have been identified.

Cloning and sequencing of the structural gene for the small subunit of methylamine dehydrogenase from Methylobacteriumextorquens AM1: Evidence for two tryptophan residues involved in the active center

In two independent clone libraries, clones were identified that hybridized with oligonucleotide probes based on N- or C-terminal polypeptide sequence of the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1. Plasmids from all clones had in common a 5.2 kb Bam HI-HindIII DNA fragment. A 0.57 kb SacII-BclI subfragment that hybridized to the oligonucleotide probes was sequenced. Nucleotide sequence analysis coincided with polypeptide sequence data in the structural part of the small subunit with a single contradiction: amino acid 17 is Asp rather than Asn. The two amino acids that are involved in the active center which had not been determined from previous polypeptide sequencing proved to be tryptophans.