Here we report the cDNA-deduced amino-acid sequence of l-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FADdependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, d-amino-acid oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl 2 , prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.

A hydrophobic sequence• n motif commo to N.hydroxylating enzymesThe first committed step in the biosynthesis of various bacterial and fur,gal siderophores (low-molecular-weight iron chelators that are produc~ in response '~o iron deficiency) of the hydroxamate type, such as aerobactin, alcaligin an6, ferrichrome, involves N-hydroxylatie a of a primary amino group. This reaction is catalyzed at the expense of NADPH by a family o[ FAD-dependent enzym ~s. Some of the siderophores act as virulence factors i. A similar reaction is carried out by a family of mammalian flavin-containing dimethylaniline monooxygenases. In the latter case, the subs~rates are tertiary and secondary diet-derived alkylamines and, as such, these enzymes play a role in the degradation of xenobiotics. Deficiency in this enzyme activi:y was recognized as the cause of the inheritable 'fish-odor syndrome' (trime:hylaminuria) which is characterized [,y an increased excretion of malodorous teimethylamine'. A BLAST sear~ch 3 in the NCBI non-redundant database (as of 5 August 1997) and seqt~ence alignment with CLUSTALX (ReL 4)and MACAW (Ref. 5) of four siderophore biosynthetic enzymes from EschericMa coil (aerA; iucD) 6,7, Pseudomonu:~ aeruginosa (pvdA) ", Bordetella b:'onchiseptica (alcA) 9 and Ustilago macdis (sid 1) 1° and about 30 sequences of flavin-containing mammalian monooxygenases (nine representati-,e sequences are shown in Fig. l) revealed three dominant areas of similarity (Fig, la, b and c). As expected, all proteins contained two nucleotide-binding folds; the N-terminal fold was assigned as the FAD and the one towards the centre as the NADP binding site ( Fig. la and b, respectively)! u2. The FAD-binding site of the mammalian monooxygenases has the typical fingerprint sequence GXGXXG, whereas the siderophore biosynthetic enzymes (alcA, iucD, pvdA and sidl, see Fig. 1) exhibit an exchange of the last glycine to proline. This quite unusual replacement is unique among FAD-dependent enzymes and it was assumed to be the cause of the weak binding of FAD to lysine N~-hydroxylase (EC 1.14.13...)L~. Similarly, alcA and pvdA possess an alanine and sidl a serine instead o[ the last glycine in the putative NADP-binding site.The third and new sequence similarity was discovered in the C-terminal part of the proteins (Fig. lc). The similarity starts with a highly conserved aspartate and is followed by eight hydrophobic amino acids. The core region consists of the sequence L/FATGY and ends with a proline after four variable amino acids. An exception was found in the two ornithine NS-hydroxylases

A nonaheme cytochrome c was purified to homogeneity from the soluble and the membrane fractions of the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex. The gene encoding for the protein was cloned and sequenced. The primary structure of the multiheme protein was highly homologous to that of the nonaheme cytochrome c from D. desulfuricans ATCC 27774 and to that of the 16-heme HmcA protein from Desulfovibrio vulgaris Hildenborough. The analysis of the sequence downstream of the gene encoding for the nonaheme cytochrome c from D. desulfuricans Essex revealed an open reading frame encoding for an HmcB homologue. This operon structure indicated the presence of an Hmc complex in D. desulfuricans Essex, with the nonaheme cytochrome c replacing the 16-heme HmcA protein found in D. vulgaris. The molecular and spectroscopic parameters of nonaheme cytochrome c from D. desulfuricans Essex in the oxidized and reduced states were analyzed. Upon reduction, the pI of the protein changed significantly from 8.25 to 5.0 when going from the Fe(III) to the Fe(II) state. Such redox-induced changes in pI have not been reported for cytochromes thus far; most likely they are the result of a conformational rearrangement of the protein structure, which was confirmed by CD spectroscopy. The reactivity of the nonaheme cytochrome c toward [Ni,Fe] hydrogenase was compared with that of the tetraheme cytochrome c(3); both the cytochrome c(3) and the periplasmic [Ni,Fe] hydrogenase originated from D. desulfuricans Essex. The nonaheme protein displayed an affinity and reactivity toward [Ni,Fe] hydrogenase [K(M) = 20.5 +/- 0.9 microM; v(max) = 660 +/- 20 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)] similar to that of cytochrome c(3) [K(M) = 12.6 +/- 0.7 microM; v(max) = 790 +/- 30 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)]. This shows that nonaheme cytochrome c is a competent physiological electron acceptor for [Ni,Fe] hydrogenase.

Here we report the cDNA-deduced amino-acid sequence of l-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FADdependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, d-amino-acid oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl 2 , prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.