The Novel Amidase PcnH Initiates the Degradation of Phenazine-1-Carboxamide in Sphingomonas histidinilytica DS-9

Abstract

Phenazines are an important class of secondary metabolites and are primarily named for their heterocyclic phenazine cores, including phenazine-1-carboxylic acid (PCA) and its derivatives, such as phenazine-1-carboxamide (PCN) and pyocyanin (PYO). Although several genes involved in the degradation of PCA and PYO have been reported so far, the genetic foundations of PCN degradation remain unknown. In this study, a PCN-degrading bacterial strain, Sphingomonas histidinilytica DS-9, was isolated. The gene pcnH, encoding a novel amidase responsible for the initial step of PCN degradation, was cloned by genome comparison and subsequent experimental validation. PcnH catalyzed the hydrolysis of the amide bond of PCN to produce PCA, which shared low identity (only 26 to 33%) with reported amidases. The K_m and k_cat values of PcnH for PCN were 33.22 ± 5.70 μM and 18.71 ± 0.52 s^-1, respectively. PcnH has an Asp-Lys-Cys motif, which is conserved among amidases of the isochorismate hydrolase-like (IHL) superfamily. The replacement of Asp37, Lys128, and Cys163 with alanine in PcnH led to the complete loss of enzymatic activity. Furthermore, the genes pcaA1A2A3A4 and pcnD were found to encode PCA 1,2-dioxygenase and 1,2-dihydroxyphenazine (2OHPC) dioxygenase, which were responsible for the subsequent degradation steps of PCN. The PCN-degradative genes were highly conserved in some bacteria of the genus Sphingomonas, with slight variations in the sequence identities. IMPORTANCE Phenazines have been widely acknowledged as a natural antibiotic for more than 150 years, but their degradation mechanisms are still not completely elucidated. Compared with the studies on the degradation mechanism of PCA and PYO, little is known regarding PCN degradation by far. Previous studies have speculated that its initial degradation step may be catalyzed by an amidase, but no further studies have been conducted. This study identified a novel amidase, PcnH, that catalyzed the hydrolysis of PCN to PCA. In addition, the PCA 1,2-dioxygenase PcaA1A2A3A4 and 2OHPC dioxygenase PcnD were also found to be involved in the subsequent degradation steps of PCN in S. histidinilytica DS-9. And the genes responsible for PCN catabolism are highly conserved in some strains of Sphingomonas. These results deepen our understanding of the PCN degradation mechanism.

Keywords: Sphingomonas histidinilytica DS-9; amidase PcnH; degradation; phenazine-1-carboxamide.

FIG 1

Utilization of PCN as the sole carbon and nitrogen source for growth by strain DS-9. □, PCN control; ■, PCN with strain DS-9; ●, cell density of strain DS-9 with PCN; ▴, PCA generated and degraded. Error bars represent the standard errors from three replicates.

FIG 2

Degradation pathway of PCN in strain DS-9. (A and B) HPLC analysis of metabolites that appeared during the degradation of PCN by strain DS-9. (C) MS analysis of compound I. The prominent protonated molecular ion of compound I was m/z 224.0817 [M+H]⁺, which was identified as phenazine-1-carboxamide (C₁₃H₉N₃O⁺; m/z 224.0818), with a −0.7-ppm error. (D) MS analysis of compound II. The molecular ion mass of compound II was m/z 225.0656 [M+H]⁺, which corresponded to the protonated derivative of phenazine-1-carboxylic acid (C₁₃H₈N₂O₂⁺; m/z 225.0658), with a −0.9-ppm error. (E) MS analysis of compound III (m/z 211.0504 [M−H]⁻). The molecular ion mass of compound III was m/z 211.0504 [M−H]⁻, which was in agreement with the deprotonated derivative of 1,2-dihydroxyphenazine (C₁₂H₈N₂O₂⁻; m/z 211.0513), with a −3.7-ppm error. A mass error between −5 ppm and 5 ppm is acceptable for the identification of compounds (27). (F) Proposed metabolic pathway of PCN in strain DS-9.

FIG 3

Identification of hydrolysis product of PCN by PcnH. (A) HPLC analysis of metabolites that appeared during the conversion of PCN by PcnH. (B) MS analysis of compound I. The molecular ion mass of compound I was m/z 224.0818 [M+H]⁺, which was consistent with the protonated derivative of phenazine-1-carboxamide (C₁₃H₉N₃O⁺; m/z 224.0818), with a 0.3-ppm error. (C) MS analysis of compound II. The molecular ion mass of compound II was m/z 225.0658 [M+H]⁺, which was consistent with the protonated derivative of phenazine-1-carboxylic acid (C₁₃H₈N₂O₂⁺; m/z 225.0658), with a 0.7-ppm error.

FIG 4

Phylogenetic analysis of PcnH with other homologous amidases from the NCBI Swiss-Prot protein database. The multiple alignment was performed using the ClustalX 2.1 software. The neighbor-joining method was used to construct the unrooted phylogenetic tree through MEGA 7.0 software. Bootstrap percentages (based on 1,000 replications) of >50% are shown on the branches. The scale bar represents 0.10 substitution per site. According to their respective amino acid sequence and function, the clustering of amidases is displayed in different colors. The deduced PcnH protein shared a low amino acid sequence identity with several biochemically characterized amidases from Pseudomonas syringae pv. syringae B728a (ureidoacrylate amidohydrolase RutB [Q4ZXR8]; 30.84% identity), Pseudomonas stutzeri A1501 (ureidoacrylate amidohydrolase RutB [A4VQH5]; 30.73% identity), Kibdelosporangium sp. strain MJ126-NF4 (N-carbamoylsarcosine amidase [CTQ96324]; 25.89% identity), Arthrobacter sp. (N-carbamoylsarcosine amidase [P32400]; 32.84% identity), and Arthrobacter sp. strain DR-2P (N-carbamoylsarcosine amidase [VII98709]; 28.64% identity). Proteins marked with asterisks are biochemically uncharacterized proteins.

FIG 5

Phylogenetic analysis of PcnD with other homologous dioxygenases from the NCBI Swiss-Prot protein database. The multiple-alignment analysis was performed using the ClustalX 2.1 software. The neighbor-joining method was used to construct the unrooted phylogenetic tree through MEGA 7.0 software. Bootstrap percentages (based on 1,000 replications) of >50% are shown on the branches. The scale bar represents 0.05 substitution per site.

FIG 6

Comparison of PCN degradation gene clusters from Sphingomonas spp.: Sphingomonas histidinilytica DS-9 (CP080430 to CP080436), S. histidinilytica UM2 (FUYM01000001 to FUYM01000059), Sphingomonas sp. strain C8-2 (CP034356 to CP034357), S. wittichii DP58 (AHKO01000001 to AHKO01000739), S. wittichii DC-6 (CP021181 to CP021192), S. wittichii YL-JM2C (ASTM01000001 to ASTM01000273), and S. wittichii MPO218 (CP059319 to CP059321).