Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action.

Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNAfMet to fMet-tRNAfMet is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH2-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using in vivo and in vitro approaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNAfMet. Dihydrofolate (DHF) formed as a by-product in the in vitro assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆fmt strain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment to Escherichia coli showed a decrease in the reduced folate species (THF, 5,10-CH2-THF, 5-CH3-THF, 5,10-CH+-THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNAfMet.

Salmonella Typhimurium outer membrane protein A (OmpA) renders protection from nitrosative stress of macrophages by maintaining the stability of bacterial outer membrane.

Bacterial porins are highly conserved outer membrane proteins used in the selective transport of charged molecules across the membrane. In addition to their significant contributions to the pathogenesis of Gram-negative bacteria, their role(s) in salmonellosis remains elusive. In this study, we investigated the role of outer membrane protein A (OmpA), one of the major outer membrane porins of Salmonella, in the pathogenesis of Salmonella Typhimurium (STM). Our study revealed that OmpA plays an important role in the intracellular virulence of Salmonella. An ompA deficient strain of Salmonella (STM ΔompA) showed compromised proliferation in macrophages. We found that the SPI-2 encoded virulence factors such as sifA and ssaV are downregulated in STM ΔompA. The poor colocalization of STM ΔompA with LAMP-1 showed that disruption of SCV facilitated its release into the cytosol of macrophages, where it was assaulted by reactive nitrogen intermediates (RNI). The enhanced recruitment of nitrotyrosine on the cytosolic population of STM ΔompAΔsifA and ΔompAΔssaV compared to STM ΔsifA and ΔssaV showed an additional role of OmpA in protecting the bacteria from host nitrosative stress. Further, we showed that the generation of greater redox burst could be responsible for enhanced sensitivity of STM ΔompA to the nitrosative stress. The expression of several other outer membrane porins such as ompC, ompD, and ompF was upregulated in STM ΔompA. We found that in the absence of ompA, the enhanced expression of ompF increased the outer membrane porosity of Salmonella and made it susceptible to in vitro and in vivo nitrosative stress. Our study illustrates a novel mechanism for the strategic utilization of OmpA by Salmonella to protect itself from the nitrosative stress of macrophages.

Metabolic Flux of N10-Formyltetrahydrofolate Plays a Critical Role in the Fidelity of Translation Initiation in Escherichia coli.

One-carbon metabolism produces methionine and N10-formyl-tetrahydrofolate (N10-fTHF) required for aminoacylation and formylation of initiator tRNA (i-tRNA), respectively. In Escherichia coli, N10-fTHF is made from 5, 10-methylene-THF by a two-step reaction using 5,10-methylene-THF dehydrogenase/cyclohydrolase (FolD). The i-tRNAs from all domains of life possess a highly conserved sequence of three consecutive G-C base pairs (3GC pairs) in their anticodon stem. A 3GC mutant i-tRNA (wherein the 3GC pairs are mutated to those found in elongator tRNAMet) is incompetent in initiation in E. coli (even though it is efficiently aminoacylated and formylated). Here, we show that E. coli strains having mutations in FolD (G122D or C58Y or P140L) allow a plasmid encoded 3GC mutant i-tRNA to participate in initiation. In vitro, the FolD mutants are highly compromised in their dehydrogenase/cyclohydrolase activities leading to reduced production of N10-fTHF and decreased rates of i-tRNA formylation. The perturbation of one-carbon metabolism by trimethoprim (inhibitor of dihydrofolate reductase) phenocopies FolD deficiency and allows initiation with the 3GC mutant i-tRNA. This study reveals an important crosstalk between one-carbon metabolism and the fidelity of translation initiation via formylation of i-tRNA, and suggests that augmentation of the age old sulfa drugs with FolD inhibitors could be an important antibacterial strategy.

Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases from Mycobacterium smegmatis Lack Flavin Coenzyme.

5,10-Methylenetetrahydrofolate reductase (MetF/MTHFR) is an essential enzyme in one-carbon metabolism for de novo biosynthesis of methionine. Our in vivo and in vitro analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR in Mycobacterium smegmatis, failed to reveal their function as MTHFRs. However, we identified two hypothetical proteins, MSMEG_6596 and MSMEG_6649, as noncanonical MTHFRs in the bacterium. MTHFRs are known to be oligomeric flavoproteins. Both MSMEG_6596 and MSMEG_6649 are monomeric proteins and lack flavin coenzymes. In vitro, the catalytic efficiency (kcat/Km ) of MSMEG_6596 (MTHFR1) for 5,10-CH2-THF and NADH was ∼13.5- and 15.3-fold higher than that of MSMEG_6649 (MTHFR2). Thus, MSMEG_6596 is the major MTHFR. This interpretation was further supported by better rescue of the E. coli Δmthfr strain by MTHFR1 than by MTHFR2. As identified by liquid chromatography-tandem mass spectrometry, the product of MTHFR1- or MTHFR2-catalyzed reactions was 5-CH3-THF. The M. smegmatis Δmsmeg_6596 strain was partially auxotrophic for methionine and grew only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the Δmsmeg_6596 strain was more sensitive to folate pathway inhibitors (sulfachloropyridazine, p-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are two noncanonical MTHFR proteins that are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.IMPORTANCE MTHFR/MetF is an essential enzyme in a one-carbon metabolic pathway for de novo biosynthesis of methionine. MTHFRs are known to be oligomeric flavoproteins. Our in vivo and in vitro analyses of Mycobacterium smegmatis MSMEG_6664/MSMEI_6484, annotated as putative MTHFR, failed to reveal their function as MTHFRs. However, we identified two of the hypothetical proteins, MSMEG_6596 and MSMEG_6649, as MTHFR1 and MTHFR2, respectively. Interestingly, both MTHFRs are monomeric and lack flavin coenzymes. M. smegmatis deleted for the major mthfr (mthfr1) was partially auxotroph for methionine and more sensitive to folate pathway inhibitors (sulfachloropyridazine, para-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are novel MTHFRs involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.

Rapid formylation of the cellular initiator tRNA population makes a crucial contribution to its exclusive participation at the step of initiation.

Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNACUA/3GC do not affect its formylation. However, the i-tRNACUA/3GC is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene (fmtam274), which affords initiation with i-tRNACUA/3GC. Replacement of fmt with fmtam274 in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNACUA/3GC into the fmtam274 strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNACUA/3GC causes a low level suppression of am274 in fmtam274. Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNACUA/3GC to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site.

Rewiring of one carbon metabolism in Salmonella serves as an excellent live vaccine against systemic salmonellosis.

Live attenuated vaccines are superior to the killed or subunit vaccines. We designed a Salmonella Typhimurium strain by deleting folD gene (encoding methylenetetrahydrofolate dehydrogenase-cyclohydrolase) in the presence of a heterologous fhs gene (encoding formyltetrahydrofolate synthetase) and tested its vaccine potential under stringent conditions of lethal and sub-lethal challenges with virulent Salmonella in the murine model. The efficacy of the vaccine in conferring protection against Salmonella infection was determined in a wide range of host conditions of systemic infection, corresponding to human young adults, neonates, geriatric age and, importantly, to the immune compromised state of pregnancy. The standardized vaccination regime comprised a primary dose of 104 CFU/animal followed by a booster dose of 102 CFU/animal on day 7. Challenge with the virulent pathogen was done at day 7 post-administration of the booster. Subsequently, the mortality, morbidity, systemic colonization, antibody response and cytokine profiling were determined. The vaccinated cohort showed a strong protection against virulent pathogen in all models tested. The serum anti-Salmonella antibody titers and cytokine levels were significantly higher in the vaccinated cohort compared to the mock vaccinated cohort. Thus, we report the development and validation of a live attenuated vaccine candidate conferring excellent protection against Salmonellosis and typhoid fever.

Utilisation of 10-formyldihydrofolate as substrate by dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) tranformylase/IMP cyclohydrolase (PurH) in Escherichia coli.

Dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (PurH) play key roles in maintaining folate pools in cells, and are targets of antimicrobial and anticancer drugs. While the activities of bacterial DHFR and PurH on their classical substrates (DHF and 10-CHO-THF, respectively) are known, their activities and kinetic properties of utilisation of 10-CHO-DHF are unknown. We have determined the kinetic properties (kcat/Km) of conversion of 10-CHO-DHF to 10-CHO-THF by DHFR, and to DHF by PurH. We show that DHFR utilises 10-CHO-DHF about one third as efficiently as it utilises DHF. The 10-CHO-DHF is also utilised (as a formyl group donor) by PurH albeit slightly less efficiently than 10-CHO-THF. The utilisation of 10-CHO-DHF by DHFR is ~50 fold more efficient than its utilisation by PurH. A folate deficient Escherichia coli (∆pabA) grows well when supplemented with adenine, glycine, thymine and methionine, the metabolites that arise from the one-carbon metabolic pathway. Notably, when the ∆pabA strain harboured a folate transporter, it grew in the presence of 10-CHO-DHF alone, suggesting that it (10-CHO-DHF) can enter one-carbon metabolic pathway to provide the required metabolites. Thus, our studies reveal that both DHFR and PurH could utilise 10-CHO-DHF for folate homeostasis in E. coli.

Species-Specific Interactions of Arr with RplK Mediate Stringent Response in Bacteria.

Bacteria respond to stressful growth conditions through a conserved phenomenon of stringent response mediated by synthesis of stress alarmones ppGpp and pppGpp [referred to as (p)ppGpp]. (p)ppGpp synthesis is known to occur by ribosome-associated RelA. In addition, a dual-function protein, SpoT (with both synthetase and hydrolase activities), maintains (p)ppGpp homeostasis. The presence of (p)ppGpp is also known to contribute to antibiotic resistance in bacteria. Mycobacterium smegmatis possesses Arr, which inactivates rifampin by its ADP ribosylation. Arr has been shown to be upregulated in response to stress. However, the roles Arr might play during growth have remained unclear. We show that Arr confers growth fitness advantage to M. smegmatis even in the absence of rifampin. Arr deficiency in M. smegmatis resulted in deficiency of biofilm formation. Further, we show that while Arr does not interact with the wild-type Escherichia coli ribosomes, it interacts with them when the E. coli ribosomal protein L11 (a stringent response regulator) is replaced with its homolog from M. smegmatis The Arr interaction with E. coli ribosomes occurs even when the N-terminal 33 amino acids of its L11 protein were replaced with the corresponding sequence of M. smegmatis L11 (Msm-EcoL11 chimeric protein). Interestingly, Arr interaction with the E. coli ribosomes harboring M. smegmatis L11 or Msm-EcoL11 results in the synthesis of ppGpp in vivo Our study shows a novel role of antibiotic resistance gene arr in stress response.IMPORTANCEMycobacterium smegmatis, like many other bacteria, possesses an ADP-ribosyltransferase, Arr, which confers resistance to the first-line antituberculosis drug, rifampin, by its ADP ribosylation. In this report, we show that in addition to its known property of conferring resistance to rifampin, Arr confers growth fitness advantage to M. smegmatis even when there is no rifampin in the growth medium. We then show that Arr establishes species-specific interactions with ribosomes through the N-terminal sequence of ribosomal protein L11 (a stringent response regulator) and results in ppGpp (stress alarmone) synthesis. Deficiency of Arr in M. smegmatis results in deficiency of biofilm formation. Arr protein is physiologically important both in conferring antibiotic resistance as well as in mediating stringent response.

Uracil DNA glycosylase (UDG) activities in Bradyrhizobium diazoefficiens: characterization of a new class of UDG with broad substrate specificity.

Repair of uracils in DNA is initiated by uracil DNA glycosylases (UDGs). Family 1 UDGs (Ung) are the most efficient and ubiquitous proteins having an exquisite specificity for uracils in DNA. Ung are characterized by motifs A (GQDPY) and B (HPSPLS) sequences. We report a novel dimeric UDG, Blr0248 (BdiUng) from Bradyrhizobium diazoefficiens. Although BdiUng contains the motif A (GQDPA), it has low sequence identity to known UDGs. BdiUng prefers single stranded DNA and excises uracil, 5-hydroxymethyl-uracil or xanthine from it. BdiUng is impervious to inhibition by AP DNA, and Ugi protein that specifically inhibits family 1 UDGs. Crystal structure of BdiUng shows similarity with the family 4 UDGs in its overall fold but with family 1 UDGs in key active site residues. However, instead of a classical motif B, BdiUng has a uniquely extended protrusion explaining the lack of Ugi inhibition. Structural and mutational analyses of BdiUng have revealed the basis for the accommodation of diverse substrates into its substrate binding pocket. Phylogenetically, BdiUng belongs to a new UDG family. Bradyrhizobium diazoefficiens presents a unique scenario where the presence of at least four families of UDGs may compensate for the absence of an efficient family 1 homologue.

Two highly conserved features of bacterial initiator tRNAs license them to pass through distinct checkpoints in translation initiation.

Eubacterial translation initiation involves assembly of tRNAfMet, mRNA, initiation factors (IFs) and 30S ribosome in a 30S pre-initiation complex (30S pre-IC), which rearranges and joins 50S ribosome to form 70S IC. Upon releasing IFs, 70S IC becomes elongation-competent 70S. The direct recruitment of initiator tRNA (tRNAfMet) into the ribosomal P-site, crucial in accurate initiation of translation, is attributed to two conserved features of tRNAfMet: (i) formylation of amino acid attached to it and, (ii) the presence of three consecutive G-C base pairs (3GC base pairs) in the anticodon stem. However, the precise roles of these two conserved features of tRNAfMet during the various steps of initiation remain unclear. Using natural and engineered tRNAs, we show that the 3GC pairs license tRNAfMet transitions from 30S to 70S IC and then to elongation-competent 70S by release of IF3. Of the 3GC pairs, the middle GC pair (G30-C40), or merely G30 (in a specific context) suffices in this role and is essential for the sustenance of Escherichia coli. Furthermore, rescue of formylase deficient E. coli by overproduced tRNAfMet reveals that the feature of formylation licenses initial targeting of tRNAfMet to 30S ribosome

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli.

Most organisms possess bifunctional FolD [5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrahdrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (EcoFolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (CpeFolD) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of CpeFolD is about five times more efficient than that of EcoFolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is ∼ 10 times more efficient than that of EcoFolD. Kinetic parameters for CpeFhs were also determined for utilization of all of its substrates. Both CpeFolD and CpeFchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of CpeFolD and CpeFchA is also necessary to rescue an E. coli folD deletion strain (harbouring CpeFhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.

Impact of Mutating the Key Residues of a Bifunctional 5,10-Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase from Escherichia coli on Its Activities.

Methylenetetrahydrofolate dehydrogenase-cyclohydrolase (FolD) catalyzes interconversion of 5,10-methylene-tetrahydrofolate and 10-formyl-tetrahydrofolate in the one-carbon metabolic pathway. In some organisms, the essential requirement of 10-formyl-tetrahydrofolate may also be fulfilled by formyltetrahydrofolate synthetase (Fhs). Recently, we developed an Escherichia coli strain in which the folD gene was deleted in the presence of Clostridium perfringens fhs (E. coli ΔfolD/p-fhs) and used it to purify FolD mutants (free from the host-encoded FolD) and determine their biological activities. Mutations in the key residues of E. coli FolD, as identified from three-dimensional structures (D121A, Q98K, K54S, Y50S, and R191E), and a genetic screen (G122D and C58Y) were generated, and the mutant proteins were purified to determine their kinetic constants. Except for the R191E and K54S mutants, others were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. While the R191E mutant showed high cyclohydrolase activity, it retained only a residual dehydrogenase activity. On the other hand, the K54S mutant lacked the cyclohydrolase activity but possessed high dehydrogenase activity. The D121A and G122D (in a loop between two helices) mutants were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. In vivo and in vitro characterization of wild-type and mutant (R191E, G122D, D121A, Q98K, C58Y, K54S, and Y50S) FolD together with three-dimensional modeling has allowed us to develop a better understanding of the mechanism for substrate binding and catalysis by E. coli FolD.

One-carbon metabolic pathway rewiring in Escherichia coli reveals an evolutionary advantage of 10-formyltetrahydrofolate synthetase (Fhs) in survival under hypoxia.

In cells, N(10)-formyltetrahydrofolate (N(10)-fTHF) is required for formylation of eubacterial/organellar initiator tRNA and purine nucleotide biosynthesis. Biosynthesis of N(10)-fTHF is catalyzed by 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). All eubacteria possess FolD, but some possess both FolD and Fhs. However, the reasons for possessing Fhs in addition to FolD have remained unclear. We used Escherichia coli, which naturally lacks fhs, as our model. We show that in E. coli, the essential function of folD could be replaced by Clostridium perfringens fhs when it was provided on a medium-copy-number plasmid or integrated as a single-copy gene in the chromosome. The fhs-supported folD deletion (ΔfolD) strains grow well in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N(10)-fTHF in the ΔfolD strain (supported by plasmid-borne fhs) were limiting despite the high capacity of the available Fhs to synthesize N(10)-fTHF in vitro. Auxotrophy for purines could be alleviated by supplementing formate to the medium, and that for glycine was alleviated by engineering THF import into the cells. The ΔfolD strain (harboring fhs on the chromosome) showed a high NADP(+)-to-NADPH ratio and hypersensitivity to trimethoprim. The presence of fhs in E. coli was disadvantageous for its aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. The computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria.

A genetic analysis of the functional interactions within Mycobacterium tuberculosis single-stranded DNA binding protein.

Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of ß1-ß1'-ß2-ß3-α-ß4-ß451-ß452-ß5 secondary structure elements. MtuSSB NTD includes an additional ß-strand (ß6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Δssb strain. However, a chimeric SSB (mß4-ß5), wherein only the terminal part of NTD (ß4-ß5 region possessing L45 loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The mß4-ß6 construct obtained by substitution of the region downstream of ß5 in mß4-ß5 SSB with the corresponding region (ß6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L45 loop and the ß6 strand of MtuSSB.

1-naphthol 2-hydroxylase from Pseudomonas sp. strain C6: purification, characterization and chemical modification studies.

1-Naphthol 2-hydroxylase (1-NH) which catalyzes the conversion of 1-naphthol to 1,2-dihydroxynaphthalene was purified to homogeneity from carbaryl-degrading Pseudomonas sp. strain C6. The enzyme was found to be a homodimer with subunit molecular weight of 66 kDa. UV, visible and fluorescence spectral properties, identification of flavin moiety by HPLC as FAD, and reconstitution of apoenzyme by FAD suggest that enzyme is FAD-dependent. 1-NH accepts electron from NADH as well as NADPH. Besides 1-naphthol (K(m), 9.1 µM), the enzyme also accepts 5-amino 1-naphthol (K(m), 6.4 µM) and 4-chloro 1-naphthol (K(m), 2.3 µM) as substrates. Enzyme showed substrate inhibition phenomenon at high concentration of 1-naphthol (K(i), 283 µM). Stoichiometric consumption of oxygen and NADH, and biochemical properties suggest that 1-NH belongs to FAD containing external flavomonooxygenase group of oxido-reductase class of enzymes. Based on biochemical and kinetic properties, 1-NH from Pseudomonas sp. strain C6 appears to be different than that reported earlier from Pseudomonas sp. strain C4. Chemical modification and protection by 1-naphthol and NADH suggest that His, Arg, Cys, Tyr and Trp are at or near the active site of 1-NH.